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A review of Limbal Stem Cell Deficiency including its etiology, pathopysiology, diagnosis, and treatment.

Limbal Stem Cell Deficiency

The corneal epithelium is a stratified squamous epithelium from which superficial terminal cells are naturally shed. Limbal stem cell deficiency (LSCD) is characterized by a loss or deficiency of the stem cells in the limbus that are vital for re-population of the corneal epithelium and to the barrier function of the limbus.[1][2] When these stem cells are lost, the corneal epithelium is unable to repair and renew itself. This results in epithelial breakdown and persistent epithelial defects, corneal conjunctivalization and neovascularization, corneal scarring, and chronic inflammation. All of these contribute to loss of corneal clarity, potential vision loss, chronic pain, photophobia, and keratoplasty failure.[2][3]

The etiologies can be genetic, acquired, or idiopathic.

Genetic:

Limbal stem cell deficiency has been associated with PAX6 gene mutations, which are also implicated in aniridia[4] and Peters Anomaly.[5] Other genetic disorders that have been reported with LSCD include ectrodactylyl-ectodermal-dysplasia-clefting syndrome[6], keratitis-ichthyosis-deafness (KID)Syndrome[7], Xeroderma Pigmentosum[8], Dominantly Inherited Keratitis[9], Turner Syndrome[3] and Dyskeratosis Congenita.[10]

Acquired:

Inflammatory:

Other causes include inflammatory insults such as those seen in Steven-Johnsons Syndrome (SJS) [11], ocular cicatricial pemphigoid[12], and graft versus host disease.[13] Chronic ocular allergy such as Vernal Keratoconjunctivitis is another reported cause[14]. Neurotrophic keratopathy, whether neuronal or ischemic, can lead to this disease as well[2], as can bullous keratopathy .[15]

Infectious:

Any infections of the corneal surface such as herpes keratitis[16] and trachoma [17] can predispose to this condition.

Traumatic/Iatrogenic:

Acquired causes also include trauma from chemical or thermal burns, and patients who have undergone prior ocular surgeries or cryotherapies at the limbus may be more susceptible.[16][18] Radiation and chemotherapy are other potential causes, and systemic[19] as well as topical chemotherapeutic medications may be sufficient to cause deficiency.[20] LSCD has also been seen with benzalkonium chloride toxicity with glaucoma medications .[21] Inappropriate contact lens use with consequent hypoxia and ocular irritation with destruction of the limbus may also contribute to both focal and total limbal stem cell deficiency.[22][23]

Tumors/Overgrowth of Other Tissue:

Ocular surface tumors are a known cause of LSCD.[2] Pterygium may also cause a focal acquired absence of limbal stem cells.[24]

Risk factors for LCSD vary according to the underlying cause, as above.

Pathology typically shows conjunctivalization of the cornea which can be indicated by the presence of goblet cells in the cornea. However, lack of goblet cells may be seen in approximately one third of patients.

Limbal stem cell deficiency (LSCD) is characterized by a loss or deficiency of stem cells which are vital for re-population of the corneal epithelium.

Corneal transparency is essential for vision, and thus the outer protective stratified corneal epithelium is under constant, rapid renewal with vigorous repair mechanisms. These mechanisms are essential as the cornea is constantly desquamating, and any trauma or loss of epithelial cells must be repaired quickly. Corneal epithelium completely regenerates every 3 to 10 days requiring constant renewal of cells.[9] The repair is essential to prevent infection and to preserve vision.

Corneal stem cells are located peripherally at the limbus in the basal cell layer, in pigmented crypts called the palisades of Vogt.[25] This pigmentation is thought to help protect the stem cells from ultraviolet light damage. In the normal cornea, renewal occurs from basal cells with centripetal migration of stem cells from the periphery.[26][27] This is a structure deeply related to the function of each cell. The stem cells and their progenitors require the vascular nutrition that is found in the stromal vasculature outside the cornea, and thus they must be at the periphery.[28]

Conversely, the cornea is an avascular structure. It must remain avascular in order to prevent vascular structures from interfering with light transmission and thus vision. The limbus plays an important role in preventing vascularization of the cornea from the conjunctiva; thus with loss of integrity of the limbus, conjunctival cells migrate to the cornea resulting in corneal neovascularization .[29][30]

Primary Prevention for LCSD varies according to the underlying cause. Contact lens overwear can be treated with cessation of lenses and frequent lubrication.[22] Traumatic causes may be avoided with the use of eye protection, for example. Treatment of systemic inflammatory disease is necessary to prevent ocular complications. Similarly, treatment of severe infections before they affect the limbal stem cells is critical to avoid damage in this area.

The diagnosis of limbal stem cell deficiency is largely made on clinical grounds. Patient history and clinical observation of corneal conjunctivalization associated with persistent epithelial defects hints strongly at limbal stem cell deficiency.[31] Loss of the limbal anatomy and irregular staining with fluorescein may also be seen.[32]

Patients usually present with pain resulting from recurrent erosions and decreased vision. Other symptoms may includecontact lens intolerance, photophobia, tearing, and blepharospasm.[16] The history will vary depending on the etiology. For example, a patient with LSCD from chemical burn or trauma will give a history of such an event.

The patient with limbal stem cell deficiency will present with recurrent epithelial erosions that leads to chronic keratitits, scarring, and calcification if untreated.[16] Delayed wound healing and corneal neovascularization occur with loss of limbal stem cells[33], and eventually a process called conjunctivalization occurs. The corneal surface will be covered by conjnuctiva-like epithelium that undergoes transformation into a cornea-like epithelium with loss of goblet cells, a process termed conjunctival transdifferentiation[27]. Patients usually suffer from recurrent erosions and decreased vision as a result of an irregular optical interface, weak tensile strength, and an incompetent barrier function.[27]

Patients present with progressive epitheliopathy with hazy, translucent epithelium extending centrally from the limbus, most commonly from the superior limbus. Epithelial staining, from punctate changes to more confluent staining, is broadest adjacent to the involved limbus and extends centripetally into the cornea to varying degrees in a whorl shape[2]. Patients often have evidence of mild to moderate tear film dysfunction, reduced tear film break-up time, or both[21]. Infectious keratitis is a common complication.[32] In late stages, superficial and deep vascularization, persistent epithelial defects leading to ulceration, melting, and perforation, fibrovascular pannus, and finally scarring, keratinization, and calcification can be seen.[34]

Eye pain and blurry vision are a common complaint in this disease as the epithelial surface breaks down. Eye irritation, contact lens intolerance, and blurred or decreased vision were the most common symptoms in one study.[21]

A diagnosis of limbal stem cell deficiency requires both clinical signs and symptoms of the disease along with cytological evidence.[30] Typical findings of conjunctival changes to the cornea adjacent to the limbus are a hallmark of the disease.[21]

Impression cytology shows conjunctivalization of cornea, and immunohistochemical markers of conjunctiva on impression cytology of the corneal surface (e.g. absence of keratin CK3) confirms the diagnosis.[35] On impression cytology, if the corneal impression is mainly acellular or contains normal corneal epithelial cells then it becomes less likely that limbal stem cell deficiency exists. However, if the impression consists of a mixture of corneal and conjunctival epithelial cells or mainly conjunctival epithelial cells then this is highly confirmative of limbal stem cell deficiency.[31]

On histopathology of the affected area, there is invasion and overgrowth of conjunctival epithelium, neovascularization, disruption of the basement membrane, and prominent inflammatory cell infiltrates.[36] Pathology typically shows conjunctivalization of the cornea which can be indicated by the presence of goblet cells in the cornea. However, lack of goblet cells may be seen in approximately one third of patients.

In vivo confocal microscopy has also been used to help diagnose LSCD. Changes may include absence of the palisades of Vogt in the affected sector, metaplastic wing and basal epithelial cells with significantly decreased basal epithelial cell density and subbasal nerve density, and replacement of normal limbal epithelium by vascular fibrotic tissues in late stages.[37]

See the figure above for the potential causes of LSCD, though any injury or loss of limbal stem cells or their niche may lead to this disease.

Management is typically symptomatic in nature early in the disease. When limbal stem cell injury is transient, sometimes termed limbal stem cell disease or limbal stem cell distress, conservative medical measures as above may be sufficient[21][31][38] However, total limbal stem cell deficiency must be surgically managed.

Medical management is aimed at restoring the limbal microenvironment with a stepwise approach based on both stopping traumatic or toxic insults to the limbus and optimizing the ocular surface by improving the tear film, controlling inflammation, and promoting differentiation of healthy epithelium.[21] This includes steps such as discontinuing contact lenses, aggressive lubrication with preservative free artificial tears, and lid hygiene or warm compresses.[22] When the surface does not respond to such treatment, nightly topical Vitamin A ointment, short-term pulse topical corticosteroids such as methylprednisolone 1%, loteprednol etabonate 0.5%, or 0.2%, or prednisolone acetate 1%, and cyclosporine 0.05%. Punctal occlusion may be performed in patients with significant aqueous tear film deficiency, and patients with rosacea may be treated with oral doxycycline.[21] Autologous serum eyedrops may stimulate healing of the corneal surface .[39] A bandage contact lens or the PROSE scleral lens is another option to optimize the health of the ocular surface.[40]

Improvement in the ocular surface may manifest as decreased pain and increased visual acuity on follow-up examinations. Progressive epitheliopathy with hazy, translucent epithelium extending centrally from the limbus may begin to regress, as may the pattern of epithelial staining with fluorescein[21] As above, if the signs and symptoms point to a true limbal stem cell deficiency that is not improving, surgery is necessary.

Prior to surgical intervention, effective assessment of tear film production and eye closure is an important prerequisite to ensure optimal surgical outcomes.[30] Resection of pannus tissue and subsequent amniotic membrane transplant may be helpful with partial or focal limbal stem cell deficiency not responding to these treatments.[41][42]

Penetrating Keratoplasty (PK) alone is not a viable option in LSCD as the donor tissue does not include limbal stem cells in such a transplant. In addition, the pre-existing corneal vascularization and inflammation increases the risk of rejection in these patients.[2] Thus, while the transplanted ocular surface will be temporarily clear, the same problems with its restoration and repair will eventually occur unless a viable source of stem cells to repair the lost cells is found.

Unilateral vs. Bilateral Disease:

Unilateral LSCD can be treated with autologous limbal stem cell transplants from unaffected eyes, and the benefit is that systemic immunosuppression is unnecessary.[30] However, the removal of stem cells from the contralateral eye risks stem cell deficiency in the donor eye. The risk of epithelial problems in the donor eye is low when less than four to six clock hours of limbal tissue and a moderate amount of conjunctiva are removed.[16] Allogeneic transplants from donor eyes are used when the disease is bilateral.[43] Living donor tissue is preferred as cadaveric donor tissue has worse outcomes when transplanted.[44]

Ex Vivo Cultivation:

To minimize loss of donor limbal tissue and the possibility of inducing LSCD in the donor eye, newer techniques use ex vivo cultivated limbal epithelial cells for transplantation. In this technique, a smaller area (generally 2mm x 2mm) of donor cells is grown in the laboratory on fibroblast culture medium or graft tissue/amniotic membrane in order to expand the donor cell population in an attempt to increase success rates and decrease epithelialization time.[45][46] Because using animal feeder cells such a fibroblasts to grow explanted cells may represent an unknown risk in the clinical transplantation of recipients with potentially undetected viral transmission, xeno-free transplants on amniotic membrane have been investigated which only use human tissues and cells.[47]

An even newer technique for unilateral disease called Simple Limbal Epithelial Transplantation (SLET) seeds donor stem cells directly on amniotic membrane placed on the ocular surface of the recipient, altogether bypassing the need for laboratory conditions of expansion.[48] These techniques may be combined with subsequent penetrating keratoplasty to further improve visual outcomes, once the limbal stem cell niche has been restored.[49]

The newest techniques for transplanting limbal stem cells involve hydrogel lenses and plasma polymer-coated contact lenses for in vivo culture and transfer of transplanted cells.[50] These are still in the testing phase in animal studies and some small human studies.

Beyond Limbal Cells:

Other options aside from keratolimbal allograft transplantation include oral mucosal epithelial transplantation. The use of keratoprostheses, such as the modified osteoodonto keratoprosthesis and the Boston Keratoprosthesis[51] are generally a last resort for total LSCD with poor surface and tear quality. Human embryonic stem cells, hair follicle, umbilical cord, and dental pulp stem cells all show potential in recreating the corneal phenotype but none has been perfected to date. [30] Each of these is an attempt to recreate the ocular surface in order to create clear vision.

Postoperative treatment consists of preservative-free topical antibiotic, topical immunosuppressants, and frequent preservative-free artificial tears. Steroids are rapidly tapered in autologous limbal transplantation.[16] Transplantation of an allograft poses a high risk of rejection even in HLA matched recipients. Therefore, graft survival depends on systemic immunosuppression for a prolonged, if not indefinite, period. .[49][52]

During the early postoperative period the limbal explant is carefully monitored for any areas of epithelial loss. Conjunctival epithelium can cross the explant at these sites and gain access to the corneal surface. If conjunctival encroachment is observed, mechanical debridement of conjunctival cells should be promptly carried out.[16]

Similarly, patients should be followed regularly for signs of graft rejection and treated appropriately. Signs of rejection include sectoral limbal injection, edema and infiltration of the graft, punctate keratopathy, and epithelial irregularities and defects, and surface keratinization.[53] [16] Risk factors for failure of a graft include blink-related microtrauma, conjunctival inflammation, increased intraocular pressure (IOP), aqueous teardeficient dry eye, lagophthalmos, and pathogenic symblepharon, all of which should be addressed at follow-up visits should they arise.[54]

Untreated limbal stem cell deficiency causes pain, decreased vision, and recurrent epithelial erosions that predispose to infection and loss of vision. Infectious keratitis is common with this disease, and patients who wear contact lenses for extended periods of time, have persistent epithelial defects, and use topical immunosuppressive medications are at increased risk.[32] After surgical treatment, there is a risk of rejection from allogeneic transplants.[49] It is possible that the cornea will not remain clear and further surgery such as repeat stem cell transplant or penetrating keratoplasty may be necessary[49]

Cultivated Oral Mucosal Epithelial Transplantation (COMET):

Patients with live related stem cell transplantation or cultivated oral mucosal epithelial transplantation (COMET) along with lamellar or penetrating keratoplasty have poor outcomes even with long-term immunosuppression.[54][55][56] The use of fibrin glue rather than amniotic membrane for COMET and optimizing the ocular surface prior to transplant improved outcomes in a recent study, and it is possible that future modifications to technique may improve these outcomes further.[57]

Cultivated Limbal Epithelial Transplantation (CLET):

Studies have shown that CLET is as effective as direct limbal transplantation for LSCD while requiring less donor tissue and thus being safer for donor eyes.[45][58][59][60][61] Studies of CLET have shown a 68-80% success rate.[62][63] In a review of outcomes of cultured limbal epithelial cell therapy published from 1997 to 2011 with data from 583 patients, the overall success rate was 76%.[60] However, this varies by The success rate of a transplant is significantly higher with an increased number of transplanted stem cells and failures tend to happen within the first year.[63]

The largest study of xeno-free explant culture transplants showed a 71% success rate in 200 recipient eyes with a mean follow-up of approximately 5 years and up to 10 years.[46][49] Supplemental corneal transplant (PK) has a survival rate of 1 year, with a median survival of 3.3 years.[49]

In a recent meta-analysis of the outcomes of keratolimbal allografting for LSCD, postoperative corrected distance visual acuity (CDVA) was 2 or more lines better than the preoperative visual acuity in 31%to 67% of eyes .[55]

Simple Limbal Epithelial Transplant:

In a study of 6 patients with total unilateral LSCD, visual acuity improved from worse than 20/200 in all recipient eyes before SLET surgery to 20/60 or better in four (66.6%) eyes, while none of the donor eyes developed any complications. Mean follow-up was 9.2 months.[48]

Boston Keratoprosthesis:

The Boston K-pro has been found to have good short-term visual and anatomical outcome in patients with bilateral LSCD[64] with vision of 20/40 or better at 6 months. One large study found a final postoperative CDVA 2 or more lines better than the preoperative visual acuity in 86% (18 of 21) of eyes and a CDVA of 20/50 or better in more than two thirds of eyes up to 3 years after surgery, though these prostheses should be used with caution in eyes with SJS and other immune causes as there is an increased retention failure rate.[51]

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