|Year : 2007 | Volume
| Issue : 2 | Page : 63-69
Corneal limbal stem cells: II. ocular surface reconstruction with limbal stem cell transplantation
Anterior Segment Division, King Khaled Eye Specialist Hospital, PO Box 7191, Riyadh 11462, Saudi Arabia
|Date of Web Publication||11-Nov-2009|
Anterior Segment Division, King Khaled Eye Specialist Hospital, PO Box 7191, Riyadh 11462
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Ocular surface reconstruction has evolved greatly in the past few decades because of increased understanding of limbal stem cell location and function, as well as improved microsurgical and stem cell culture techniques. Proper diagnoses of corneal limbal stem cell dysfunction, provision of appropriate presurgical intervention and management, logical selection of the appropriate limbal stem cell transplantation technique, optimum postoperative management, and performance of lamellar or penetrating keratoplasty, when indicated, are essential steps to maximize the prognosis for graft survival and visual outcome in eyes with limbal stem cell deficiency.
Keywords: corneal limbal stem cells, ex vivo expansion, limbal stem cell deficiency, limbal stem cell transplantation
|How to cite this article:|
Jastaneiah S. Corneal limbal stem cells: II. ocular surface reconstruction with limbal stem cell transplantation. Middle East Afr J Ophthalmol 2007;14:63-9
|How to cite this URL:|
Jastaneiah S. Corneal limbal stem cells: II. ocular surface reconstruction with limbal stem cell transplantation. Middle East Afr J Ophthalmol [serial online] 2007 [cited 2020 May 27];14:63-9. Available from: http://www.meajo.org/text.asp?2007/14/2/63/57709
The term limbal stem cell deficiency (LSCD) is used to describe poor corneal limbal stem cell (SC) function caused by partial or complete destruction. The classical appearance is the triad of persistent epitheliopathy, recurrent and persistent epithelial defects, and progressive vascularization and pannus formation. ,
Ocular surface reconstruction (OSR) describes a group of surgical procedures required to correct both limbal stem cell dysfunction and mechanical abnormalities of the eyelids and conjunctiva in an effort to restore proper anatomy and function of the ocular surface and improve visual function. Technically speaking, OSR has passed through many difficult stages over the years but has recently regained its popularity. This increasing popularity and success of OCR can be attributed to 4 major breakthroughs: (1) identification of the anatomic location of corneal stem cells in the limbus; (2) advances in microsurgical techniques; (3) use of the amniotic membrane (AM) as a basement membrane and as a provider of other physiologic functions; and (4) realization of the physiologic component of tears and its importance in ocular surface integrity. ,
Surgical reconstruction of ocular surface disease started in 1977 when Thoft described conjunctival transplantation as a means of treating unilateral chemical and thermal injuries. , Unfortunately, conjunctival transdifferentiation did not produce the desired corneal phenotype as expected. Consequently, the procedure was later modified to include limbal stem cells in an effort to restore proper corneal epithelial function. In 1984, Thoft initially proposed keratoepithelioplasty for this purpose; however, it did not yield satisfactory results because of recipients' rejection of cadaver stem cells and technical difficulties associated with performing this procedure. In 1989, Kenyon and Tseng performed the first human conjunctival limbal autograft in eyes with severe ocular surface disorders and reported successful results in 26 patients. , Their work is considered the leading breakthrough in strategies for restoration of limbal stem cell function in eyes with LSCD.
Selecting the proper approach to the surgical management of LSCD depends upon proper categorization of the disorder. There are 2 categories of LSCD.  The first category includes patients with a clear history of an insult causing limbal stem cell destruction, such as chemical or thermal burns. In these cases, stem cell replacement is a prerequisite for anatomic and functional success. The second category encompasses patients with no clear history of an external insult but with a gradual loss of limbal stem cell function, such as in aniridia or ocular cicatricial pemphigoid. In these cases, stem cell replacement alone is insufficient for a complete cure; thus, treatment must be directed toward applicable developmental, hormonal, neuronal, vascular, and inflammatory factors in the limbal stroma, as well as tear deficiency syndromes and anatomic abnormalities of the eyelids and conjunctiva. ,
The management algorithm of LSCD depends on whether the condition is unilateral or bilateral, and whether it involves total or partial stem cell involvement. ,, In each case, the acute insult or underlying condition should be properly managed, particularly with respect to control of ocular surface inflammation. After initial management, restoration of limbal stem cells can be undertaken, as per the algorithm. If necessary, penetrating keratoplasty (PKP) can be performed for visual rehabilitation if rehabilitation of the ocular surface alone is insufficient in achieving this objective. Failure to stabilize the corneal epithelial surface prior to lamellar or penetrating keratoplasty is the most common cause of graft failure in these cases. ,
Limbal Stem Cell Transplantation
In unilateral or asymmetric cases of LSCD, it is preferable to harvest limbal stem cells from the uninjured or less injured fellow eye, in which case the procedure is called conjunctival limbal autograft (CLAU) transplantation. In bilateral cases of LSCD, it is necessary to harvest limbal stem cells from a living relative or cadaver, in which case the procedures are called living-related conjunctival limbal allograft (lr-CLAL) and keratolimbal allograft (KLAL) transplantation, respectively.
The techniques of CLAU and lr-CLAL carry the risk of damage to the healthy stem cells from the donor, and, hence, they may be exposed to problems of LSCD in the future.  Recent development of ex vivo expanded stem cells may solve this problem because only a small piece of stem cell containing tissue must be surgically removed from the donor eye, after which the stem cells are expanded in culture and then subsequently transferred to the injured eye.
Conjunctival Limbal Autograft (CLAU)
This technique involves the removal of two 3 x 10-mm limbal tissue grafts that contain approximately 0.5 mm of clear cornea centrally and approximately 2.5 mm of bulbar conjunctiva from the donor eye [Figure 1]. The grafts are marked before the dissection to ensure appropriate sizing and graft orientation [Figure 1]A. The grafts are usually harvested from the 1200 and 600 meridians, rather than in the exposed interpalpebral fissure, and extend for approximately 60Ί or 3 clock hours [Figure 1]B-D. Compared to the nasal and temporal meridians, stem cell populations are greater in the superior and inferior quadrants of the cornea. 
If the recipient eye is not vascularized, a 360-degree peritomy is performed at the limbus, and the conjunctiva is recessed to provide space for insertion of the donor grafts. If the recipient eye is vascularized, the 360-degree peritomy is performed 5 mm posterior to the limbus, followed by surgical removal of the limbal conjunctiva and corneal pannus by blunt superficial keratectomy [Figure 1]E.
The autografts are then transferred to the preprepared recipient eye [Figure 1]F, and secured in place with 10-0 nylon sutures to stabilize the corneal border and with 7-0 vicryl sutures for the conjunctival border [Figure 1]G. A soft contact lens or amniotic membrane may be placed on the eye, or a marginal tarsorrhaphy may be performed, to protect the graft from the shearing forces of the eyelid in the postoperative period.
If necessary, lamellar or penetrating keratoplasty can then follow the limbal transplantation procedure after a period of 3 to 12 months. , Although controversial, some authors have advocated simultaneous transplantation of limbal stem cells and keratoplasty in the late management of severe chemical injuries. 
Postoperatively, aggressive lubrication with unpreserved lubricants and/or autologous serum is mandatory. Topical steroids should be used frequently in the early postoperative course and tapered slowly over a 3-to-6 month period. If available, preservative-free preparations are preferred. Prophylactic antibiotics are required until reepithelialization is complete. Amniotic membrane transplantation may be performed to facilitate postoperative reepithelialization. 
Ipsilateral limbal autograft translocation is an option for the treatment of partial limbal deficiency.  This procedure is the same as that used when performing the conjunctival limbal autograft except that one portion from the healthy limbus in the opposite meridian of the damaged limbus is removed. This process involves removing the stem cell graft from the healthy portion of the limbus and transferring it to the deficient part in the same eye.
Living-Related Conjunctival Limbal Allograft (lr-CLAL) Transplantation
This procedure is a good option for bilateral cases of LSCD or in one-eyed patients with total LSCD. ,,, The 2 major concerns with allograft transplantation are the consequences of harvesting the tissue from the healthy donor eye and the potential adverse effects of the immunosuppressive therapy that is inevitably required in these cases.  Like CLAU, lamellar or penetrating keratoplasty can be subsequently performed if needed for visual rehabilitation.
Technically, this procedure is the same as that used when performing CLAU, with the exception that the donor tissue is harvested from a healthy eye of a living relative without transmissible diseases, such as hepatitis or acquired immune deficiency syndrome (AIDS).  The best HLA and ABO blood group compatibility is from a sibling, parent, or offspring, where matching is approximately 40%, 30%, and 30%, respectively. 
Despite the close HLA and ABO blood group match of using immediate relatives, a substantial risk of rejection of the transplanted stem cells exists. The limbal area is heavily loaded with many antigen-presenting cells, such as the Langerhans cells, which can promote rejection.  Thus the recipient patient must be immunosuppressed for a minimum of 2 years, if not indefinitely. ,,,
The postoperative regimen is identical to that of CLAU, with the exception that immunosuppressive therapy is required. ,, Initially, high doses of corticosteroids are used while awaiting full immunosuppression with cyclosporine and azathioprine, after which they may be tapered and even discontinued. Cyclosporine A, with a proposed initial daily dose of 8 to12 mg/kg of body weight, is started after the allograft transplant and is given for a week to reach a therapeutic blood level of 500 to 800 IU/mL; after which, the dose is tapered slowly over a few weeks to 4 to 5 mg/kg of body weight. After 3 months, long-term cyclosporine maintenance with 2 to 3 mg/kg/day is recommended. The initial azathioprine dose is 1.5 to 2.0 mg/kg, and the maintenance dose is 0.75 to1.0 mg/kg. Complete blood counts, as well as liver and renal function enzymes, should be monitored as long as the patient is on azathioprine and cyclosporine A. Blood levels of cyclosporine A are to be measured weekly for the first 2 months, and monthly thereafter.
The high success rate of this procedure that has been reported in several series has been attributed to the aggressive postoperative use of systemic immunosuppressive medications. ,, Despite immunosuppressive therapy, rejection rates between 25% and 33% have been reported, with a peak incidence 11 to 25 months postoperatively.  In some cases, rejection can be reversed by reinstituting high-dose systemic steroids and by increasing the dose of cyclosporine A.
Keratolimbal Allograft (KLAL) Transplantation
This procedure is an allograft transplantation of limbo-corneal tissue from a cadaver rather than from a live-related donor eye. ,,, Like lr-CLAL, it is performed for bilateral cases of LSCD or in one-eyed patients with total LSCD. This technique is essentially the same as the one used in autograft transplantation except that the grafts are from cadaver eyes, which offer the advantage of access to larger stem cell grafts but the disadvantage of a higher risk of rejection. Like CLAU and lr-CLAU, lamellar or penetrating keratoplasty can be performed for visual rehabilitation, if necessary. Solomon et al  found that subsequent keratoplasty had a better prognosis for survival and better visual outcome than concomitant keratoplasty during the first 3 postoperative years, but not thereafter.
Freehand- and microkeratome-based techniques require the use of a whole globe to harvest the graft. The stability of the donor cadaver eye and corneoscleral buttons remains a problem during dissection. ,, New techniques have been developed to address this problem. For example, cyanoacrylate tissue adhesive can be applied to the posterior side of a corneoscleral rim trephined from a specimen so that it can be secured to a disposable acrylic sphere that is attached to a cylindrical base. Performing this technique provides stability while harvesting the graft, hence minimizing the trauma to the epithelial stem cells and preserving the central corneal button.  More recently, a device consisting of a pedestal with a convex surface mounted to a flat platform has been described.  The corneoscleral button is secured by suction to maintain the required stability during harvesting.
Espana et al  studied the phenotypic outcome of KLAL-amniotic membrane graft transplantation. The transplant was used to reconstruct the corneal epithelium and was studied using immunostaining.  Seven months after the transplantation, the obtained corneal button during PKP was shown to have 5 to 6 layers of stratified epithelium resting on an eosinophilic basement membrane and on a vascular stroma. Immunostaining confirmed that the epithelial phenotype was of corneal origin.
The postoperative regimen is identical to that of lr-CLAL, with the exception that more aggressive immunosuppressive therapy may be necessary to prevent rejection. ,, Successful use of mycophenolate mofetil (CellCept) in these cases has been reported. , However, the prognosis remains guarded. ,
Ex Vivo Expansion of Limbal Stem Cells
Ex vivo expansion of limbal stem cells is a rapidly developing procedure. ,,,
In vitro propagation of epithelial cells for transplantation was first studied in 1993, and the results supported both the concept of corneal epithelial stem cell location at the limbus and the possibility of culturing epithelial cells to be used as grafts to restore the corneal epithelium after severe ocular surface injury. 
This procedure has gained increasing popularity over the classical method of CLAU and lr-CLAL because it minimizes the presumed risk of developing LSCD in the donor eye, although pathologic damage has never been reported in humans. Another advantage of this procedure over the allograft transplantation (KLAL, lr-CLAU) is the theoretical presumption that only epithelial cells are transplanted and that the antigen-presenting Langerhans cells are eliminated during the expansion. 
The technique involves bioengineering a corneal surface replacement using ex vivo expanded cultured corneal epithelial cells, which is achieved by dissecting a small piece of a stem cell bearing limbal tissue and cultivating it as an explant or as a cell suspension by using different modified techniques. It is then transplanted as an epithelial sheet to the recipient eye. The donor eye can still be either an autograft  from the fellow uninjured eye, or an allograft  from a live-related or cadaver eye.
Explant culture technique
The explant culture technique was first demonstrated by Pellegrini et al  in 1997. This technique , involves taking a 1-mm 2 biopsy sample from the limbus of the donor cornea in the same way as described in the CLAU transplantation procedure, except that a smaller piece of limbal tissue is excised. Biopsy samples were minced and treated with trypsin at 37ΊC for 3 hours. Cells were then plated in lethally irradiated (gamma-irradiated) (6000R) 3T3-J2 cells and cultured in 5% CO 2 in supplemented Dulbecco-Vogt Eagle's and Ham's F12 media. Grafts were prepared (16 days and 19 days after biopsy samples were taken) from confluent secondary cultures, which were released from the plastic dish with the neutral protease dispase II. The grafts were mounted on either petroleum gauze or a soft contact lens, with the basal layer exposed on the concave part of the contact lens. The cultured cells were then transplanted to the recipient cornea with a hydrophilic contact lens on top for protection if a contact lens was not used for mounting the graft. Corneal surface reconstruction with this procedure was effective in eyes with a total loss of limbal epithelial cells. 
Cell-suspension culture technique
Another culture technique utilizes the cell-suspension system to explant cultured corneal epithelial cells on an AM carrier. The cell-suspension method, which was used as another method of culturing epithelial cells, was compared to the explant culture method.  The cell-suspension method involves dissociating the limbal epithelial cells by dispase and then seeding them on a denuded AM. In comparison, the explant process involves preparation of the AM and the 3T3 fibroblast cells. The limbal ring is cut into 2 or 3 pieces, incubated at 37ΊC for 1 hour with 1.2 IU dispase. The limbal and stem cells are suspended in 3 mL of medium, seeded onto denuded AM spread on the bottom of culture inserts, and cocultured with mitomycin C (MMC)-inactivated 3T3 fibroblasts. The cultures were then exposed to air (air lifting) for 2 weeks to promote corneal epithelial differentiation. The culture media used was supplemented Dulbecco-Vogt Eagle's and Ham's F12 media (1:1 mixture). This culture technique claims to produce morphologically superior epithelium compared to explanted cultures.
Fibrin culture technique
The fibrin culture technique is a process where stem cells are obtained from the limbus of the contralateral healthy eye and cultivated onto a fibrin substrate.  Fibrin cultures were later grafted onto damaged corneas. The success of the cultures was measured by corneal reepithelialization and by the regression of inflammation and vascularization.
Amniotic membrane culture and transfer technique
An amniotic membrane consists of an epithelial monolayer, a thick basement membrane, and a vascular stroma. Amniotic membranes are transparent and nonantigenic; gradually resorb in vivo, facilitate reepithelialization without allowing fibrovascular growth, support epithelial differentiation, have a stimulatory effect on cell proliferation, contain extracellular matrix components that resemble those of conjunctival basement membrane cell membrane-associated molecules, and cytokine facilitate dialogues. ,,
Amniotic membrane transplantation (AMT) for ocular surface reconstruction is a promising procedure that was first reported in 1994 as a means of repairing conjunctival symblepharon and defects.  Later, when the ocular surface was maintained, successful corneal reconstruction was achieved in 40% of experimental rabbit models.  In eyes with partial limbal stem cell deficiency, transplantation of AMT alone can restore damaged corneal surfaces by aiding the expansion of residual limbal stem cells in vivo. ,
Using the AM for culturing provides a natural substrate on which the cultured limbal epithelial cells can survive and proliferate, and also simplifies the handling and structuring of the cultured cells, thereby reducing the risk of infection. ,,, Limbal epithelial explants of 1 x 2 mm are excised and cultured the same way as previously described, except that they are inoculated onto the basement membrane side of the AM.  After maintaining the culture for 2 to 3 weeks (during which time the medium has been changed every 2 days), the epithelial cell layer sheets of 2 to 3 cm are then transplanted into a denuded, preprepared recipient ocular bed.
Using EDTA-treated (denuded) AM substrate to cultivate epithelial cells was found to be better than using cellular AM (with amniotic epithelial cells). ,, Denudation of the devitalized amniotic epithelium to expose the basement membrane might be a microenvironmental promoter of transient amplifying cell (TAC) differentiation.  The proliferation activity of the ex vivo expanded human limbal cells and the overall growth rate were significantly higher on denuded AM than on intact AM. , On the other hand, the intact AM has growth factors that are potentially involved in the epithelium-stroma interaction occurring on the human ocular surface. 
Limbal stem cells are characterized by a slow cell cycle, and the lack of expression of K3 and connexin 43 (Cx43); this characteristic was used to investigate the proliferate activity of limbal stem cells expanded on AM.  The results supported the theory that intact AM preferentially preserves and expands stem cell-containing limbal basal epithelial cells by serving as a substrate that mimics their microenvironmental niche. The expanded cells comprise a Cx43-negative, K3-negative, and gap junction intercellular communication (GJIC)-deficient limbal epithelium, which are characteristics of limbal stem cells. 
The AM also has a basement membrane and an extracellular matrix system that provides the grafts with stability, which makes them easy to handle and protects them from the shearing forces on the recipient ocular surface.  Performing an allograft transplantation of corneal stem cells cultured on AM has been shown to improve ocular surface pathology resulting from chemical burns; this improvement was determined by examining in vitro the proliferation and differentiation of corneal stem cells based on their colony forming efficiency and immunohistochimestry.  Denuded AM was also successfully used as a carrier to allocultivated epithelial cells in eyes with acute and chronic Stevens-Johnson syndrome More Details (SJS), acute and chronic chemical traumas, ocular cicatricial pemphigoid, and pseudo-ocular cicatricial pemphigoid. 
In conclusion, limbal stem cell transplantation is an effective technique for restoration of a phenotypically normal corneal epithelium in eyes with LSCD. Whereas relatively straightforward limbal autograft techniques can be employed in eyes with unilateral or asymmetric LSCD, allograft transplantation from a living relative or a cadaver is required for bilateral injuries.
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