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Year : 2000  |  Volume : 48  |  Issue : 2  |  Page : 79-81

Significant impact of limbal epithelial stem cells.

Correspondence Address:
S C Tseng

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Source of Support: None, Conflict of Interest: None

PMID: 11116518

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Keywords: Cell Transplantation, Corneal Diseases, diagnosis, etiology, surgery, Epithelium, Corneal, pathology, transplantation, Humans, Limbus Corneae, pathology, Stem Cell Transplantation,

How to cite this article:
Tseng S C. Significant impact of limbal epithelial stem cells. Indian J Ophthalmol 2000;48:79-81

How to cite this URL:
Tseng S C. Significant impact of limbal epithelial stem cells. Indian J Ophthalmol [serial online] 2000 [cited 2023 Sep 23];48:79-81. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?2000/48/2/79/14881

The corneal epithelium is known to have a rapid turnover rate. For years, researchers have been intrigued by its self-renewing capacity, which undoubtedly plays an important role in maintaining corneal surface health. Through a number of experiments, the late Professor Richard Thoft[1] speculated that the loss of epithelial cells through attrition and desquamation from the central cornea would have to be replenished by migration and proliferation of epithelial progenitor cells from the peripheral cornea. Although a few earlier reports implicated the limbus as the source of these progenitor cells, the term "stem cells" had not been used or proven until researchers from Tung-Tien Sun's laboratory[2] in 1986 unequivocally demonstrated the limbal location of corneal epithelial stem cells based on the expression pattern of the cornea-specific keratin K3. This important finding marked a major advance in corneal epithelial biology. In the lead article of this issue, Dua et al[3] have succinctly reviewed the voluminous literature that has since been published on this subject. They provide a detailed account of the concept of limbal epithelial stem cells, corneal diseases characterized by limbal stem cell deficiency, and various surgical procedures for transplanting limbal epithelial stem cells.

The knowledge of limbal location of corneal epithelial stem cells, helps us understand why the conjunctival epithelium does not transdifferentiate into the corneal epithelium, and why conjunctival transplantation cannot reconstruct a corneal surface that has been severely damaged. This concept not only helps resolve other experimental paradoxes but also help clinicians understand why the limbus is the predominant site for ocular surface dysplasia or any neoplasia to develop, and why epithelial rejection seldom occurs one year after penetrating keratoplasty.

Although the term "stem cells" is not foreign to most of us, few can precisely describe the salient features that are elegantly summarized by Dua et al[3] about stem cells in general and limbal epithelial stem cells in particular. Among them, one important feature is that stem cells comprise only a fraction of the proliferative pool, and can be distinguished from their immediate offspring, transient amplifying cells, in this pool by the mitotic machinery utilized. For example, stem cells are normally slow cycling; they take a long time to complete a cell cycle, and thus are less apt to develop genetic errors during DNA synthesis. As limbal epithelial stem cells are the ultimate source of corneal epithelial regeneration, they have to be activated to produce a large population of transient amplifying cells in the event of wound healing. During each mitotic cycle, stem cells must decide whether they should be differentiated into transient amplifying cells that will naturally deplete the stem cell population, or be kept undifferentiated as stem cells so that the stem cell population may be maintained. How stem cells balance their act between these two conflicting demands is an ultimate secret dealing with how the normal corneal epithelial health is maintained.

The other salient feature of stem cells is longevity, which is inherent to the microenvironmental niche in which stem cells reside. Future studies are needed to explore the mechanism by which limbal epithelial stem cells are regulated by this unique limbal stromal environment.[4] These studies should open an entirely different realm of knowledge and help us design important new therapies. The fact that stem cells and transient amplifying cells are anatomically separable makes the ocular surface the most ideal model system to investigate these questions related to epithelial stem cells.

Inasmuch as limbal epithelial stem cells control corneal epithelial health, their deficiency can be found in a number of corneal diseases. These diseases carry several histopathologic features, among which the hallmark is "conjunctivalization", that is, invasion of conjunctival epithelial cells onto the corneal surface. In addition, the basement membrane is invariably destroyed, and the superficial corneal stroma is vascularized and infiltrated with inflammatory cells. We first defined and described the term "limbal stem cell deficiency" and these histopathologic features based on a series of animal experiments. Clinically, it is important to confirm the diagnosis of limbal stem cell deficiency in patients suffering from annoying photophobia and reduced vision while their corneas may present with some of these suspicious signs. This is because conventional corneal transplantation will have limited success. Furthermore, because some of these signs can also be found in other types of corneal diseases, patients, if wrongly diagnosed with limbal deficiency, might be subjected to the unnecessary risks of transplanting limbal epithelial stem cells. That is why we have proposed the use of impression cytology to detect the hallmark of conjunctivalization for diagnosing limbal stem cell deficiency.[5] A number of corneal diseases have since been found to exhibit cytological evidence of limbal stem cell deficiency. We believe this list will grow as we pay more attention to the status of the limbus when dealing with patients presenting with an abnormal corneal surface and compromised healing. One important bit of information we obtain from this list of corneal diseases is that pathologic processes which either destroy the limbal stem cell population or damage its stroma may eventually lead to limbal stem cell deficiency. Among all pathologic insults, chronic inflammation at the limbus appears to be a common denominator. Future studies are under way to explore the molecular mechanisms to explain how inflammation may shorten the life span of limbal epithelial stem cells. They may not only provide pathogenic insight into these diseases but also help us to devise new therapies to restore the health of limbal epithelial stem cells.

When the limbal-deficient status of the patient deteriorates, annoying photophobia and reduced vision are frequent complaints. Dua et al[3] have reviewed and summarized a list of surgical procedures for transplanting limbal epithelial stem cells. In general, selection of these procedures depends on the severity of limbal stem cell deficiency. When limbal stem cell deficiency is focal and unilateral, transplantation of autologous limbal epithelial stem cells is the treatment of choice. Nevertheless, this procedure is not without risk. In a rabbit model, we have shown that donor eyes with such large limbal removal may develop limbal deficiency if the central corneal epithelium is subsequently removed. A recent report from India[6] has also shown that progressive pseudopterygium can develop in such donor eyes. When limbal stem cell deficiency is diffuse and bilateral, transplantation of allogeneic limbal epithelial stem cells from cadaveric or living-related donors becomes indicated. The major problem encountered is the failure of limbal allografts. The failure may in part be explained by the severity of the ocular surface damage, which significantly hampers normal ocular surface defense. Therefore, prior to any grafting procedure, it is important to restore ocular surface defense by such measures as punctal occlusion, application of autologous serum, silicon-based scleral contact lens, plastic correction of lid and lash problems, and mucous membrane grafting. Without correcting these problems, transplantation of limbal epithelial stem cells may be contraindicated. The failure may also in part be due to allograft rejection, which can be subclinical and progressive despite the continuous use of systemic cyclosporin A.[7] Future studies are needed to determine if other immunosuppressive agents such as FK506 and mycophenolate mofetil may help decrease allograft rejection rates.

Another strategy to improve the survival of limbal grafts is to restore the limbal stromal environment by amniotic membrane transplantation. The amniotic membrane is the innermost layer of the placenta, and consists of a thick basement membrane and an avascular stroma. After appropriate procurement, processing, and preservation, human amniotic membrane can be used as a graft to restore damaged stroma. Although amniotic membrane was used in the early part of the last century, its ophthalmic use was reintroduced by Kim and Tseng[8] in 1995 for treating rabbits with total limbal stem cell deficiency. Since then there has been a surge of interest in using this membrane for corneal and conjunctival surface reconstruction. Up to now these clinical studies have shown that amniotic membrane transplantation facilitates epithelialization, maintains a normal epithelial phenotype, and reduces stromal inflammation, vascularization and scarring. Collectively, these actions are very desirable for restoring the limbal stromal environment. That is why we[9] and Tsubota's group in Japan[10] have reported its use in conjunction with transplantation of allogeneic limbal tissue for treating patients with bilateral and diffuse limbal stem cell deficiency.

Recently, basic research from our laboratory and elsewhere has demonstrated that amniotic membrane is an ideal substrate to preserve and expand limbal epithelial stem cells in culture. Such ex-vivo expanded limbal epithelial stem cells together with the amniotic membrane has first been shown by Tsai et al[11] from Taiwan as a limbal equivalent for reconstructing the corneal surface with limbal stem cell deficiency. Since only a small biopsy is removed from the donor eye, it obviates the unnecessary risk of large limbal removal from a normal healthy eye. Because only epithelial cells are eventually transplanted without the inclusion of other mesenchymal components in this new approach, the chance of allograft rejection may be lowered.

In summary, the concept of limbal location of corneal epithelial cells has revolutionized our understanding and therapeutic approaches for treating patients with difficult corneal surface disorders for the last 15 years. It is conceivable that clinical and basic research will continue to explore how stem cell functions can be modulated by soluble cytokines and insoluble matrix. As a result, in this new millennium we may begin to engineer severely damaged corneal surfaces in appropriately specific ways.

  References Top

Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci.1983;24:1442-43.  Back to cited text no. 1
Schermer A, Galvin S, Sun T-T. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49-62.  Back to cited text no. 2
Dua HS, Saini TS, Azuara-Blanco A, Gupta P Limbal stem cell deficiency: Concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol 2000;48:483-91.  Back to cited text no. 3
Tseng SCG. Regulation and clinical implications of corneal epithelial stem cells. Mol Biol Rep. 1996;23:47-58.  Back to cited text no. 4
Puangsricharern V, Tseng SCG. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:l476-85.  Back to cited text no. 5
Basti S, Mathur U. Unusual intermediate-term outcome in three cases of limbal autograft transplantation. Ophthalmology 1999;106:958-63.  Back to cited text no. 6
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 Eng J Med. 1999;340:1697-703.  Back to cited text no. 7
Kim JC, Tseng SCG. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995;14:473-84.  Back to cited text no. 8
Tseng SCG, 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. 9
Tsubota K, Satake Y, Ohyama M, Toda I, Takano Y, Ono M, et al. Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am J Ophthaimol 1996;122:38-52.  Back to cited text no. 10
Tsai RJF. Corneal surface reconstruction by amniotic membrane with cultivated autologous limbo-corneal epithelium. Invest Ophthalmol Vis Sci 1998;39:S429.  Back to cited text no. 11

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