LIMBAL STEM CELL DEFICIENCY

Corneal epithelial stem cells are located in the basal layers of the limbus and are essential for the renewal of the epithelium and the integrity of the corneal stroma. Pathologies/injuries affecting the limbus lead to LSCD, which can be caused either by inherited pathologies (aniridia, KID and EEC syndrome, etc.) or, more commonly, are the result of acquired factors, such as chemical/thermal injuries, ultraviolet and ionizing radiations, contact lens keratopathy, limbal surgery and conditions like Stevens-Johnson syndrome. When LSCD occurs, the neighbouring conjunctival epithelium, which is normally prevented from encroaching on the corneal surface by LSCs, migrates over the stroma. This process is known as conjunctivalization and usually is accompanied by neovascularization and abnormal fibrovascular tissue covering the corneal surface (pannus). This eventually leads to chronic inflammation, corneal opacity and vision impairment. Conventional corneal transplantation is not feasible as, in order to succeed, it requires the gradual replacement of the donor’s corneal epithelium with the recipient’s. LSCD, instead, allows/stimulates conjunctival cell ingrowth with accompanying neovascularization and inflammation, resulting in cornea graft failure. Patients with total LSCD are therefore poor candidates for corneal transplantation. In 1989, recognition of the need to replenish the stem cell population in LSCD led to the development of limbal grafts as a means of restoring corneal epithelial stem cells in the eyes of patients with LSCD. However, one serious limitation is the requirement for a sizeable limbal donation, as up to 30-40% must be harvested from the contralateral donor eye. This may harm the structural integrity of the remaining healthy eye, and prevents the repetition of the procedure in case of failure. For these reasons, in the last 10-15 years, ex vivo expansion of LSCs has been pursued. In 1997, Pellegrini et al described the successful reconstruction of the ocular surface in 2 patients with severe LSCD using transplantation of corneal epithelial stem cells isolated from 1mm2 limbal biopsies and expanded in vitro (Pellegrini et al., Lancet 1997; 349: 990-3). Many other studies describing the clinical application of LSCs expanded in vitro have since been reported, with success rates ranging from 46 to 100% and follow-up times from 6 months to 10 years (Ferrari et al., Exp Rev Ophthalmol 2009; 4: 317-29). In 2002, FBOV established a Cell Factory which has since distributed corneal stem cell grafts to nearly 20 public and private hospitals throughout Italy with 166 patients with unilateral LSCD treated (Di Iorio et al. Ocular Surface 2010; 8: 146-153). In more than 70% of patients, grafting of autologous-cultured LSCs has led to the disappearance of clinical symptoms and long-term restoration of a clear and stable corneal epithelium.

 

While the above described therapeutic strategies are well developed for patients with unilateral LSCD, treatments for patients with bilateral LSCD are still under scrutiny. Limbal allografts were initially performed using tissue from a living relative donor with healthy eyes (living-related limbal allograft) or a deceased donor (keratolimbal allograft transplantation). However, due to the vascularity of the limbus, limbal allografts are at higher risks of rejection compared to conventional corneal transplantation. The antigenic challenge to the host is higher in limbal transplanted tissues because of their higher number of Langerhans cells. For these reasons, recipients require long-term systemic immunosuppression (prednisone, cyclosporine, etc) together with topical steroids to prolong graft survival. Several studies have confirmed the importance of systemic immunosuppression in patients undergoing limbal allograft. One study found 87% of patients that were treated with systemic immunosuppression had a stable ocular surface compared to 62% that were not treated. Systemic side effects of immunosuppression include severe adverse events such as death, myocardial infarction, cerebrovascular event, and secondary tumours. Minor adverse events of immunosuppression include alterations in biochemistry, increased cardiovascular risk factors, and infections requiring hospitalization. In a large retrospective series of 136 patients undergoing LSC transplantation and systemic immunosuppression with a mean follow-up of 4,5 years, 1,5% had a severe adverse event and 15,4% had minor adverse events. Similar considerations are also true for transplantation of donor-derived cultured LSCs. For this reason transplantation of autologous stem cells from sources which are alternative to the limbus, would be an interesting and promising new approach. Some investigators have recently proposed the use of embryonic stem cells, bone marrow-derived mesenchymal stem cells and iPS. Results have so far been obtained only in animal models of corneal damage and raise some concern as these cells are not of “cornea” or “limbus” origin and could therefore lead to the development of atypical cell lineages. A more interesting approach would therefore be to use cells of ocular origin (conjunctival epithelial cells) or from tissues (oral mucosa) that are routinely used by ophthalmic surgeons.

 

A few studies have reported the transplantation of ex vivo cultured oral mucosal epithelial cells for the treatment of patients with bilateral LSCD. The rationale is that transplantation of oral mucosa to the eye is a well-documented surgical procedure in ophthalmology. Inatomi et al showed that corneal clarity, improved visual acuity and stable corneal surface persisted in 67% of the patients (Inatomi et al, Am J Ophthalmol 2006; 141: 267-75 and 142: 757-64). However, oral mucosa is a non-ocular tissue and thus retains the characteristics of the tissue of origin. Opacity at longer follow-up times and peripheral neovascularization have, in fact, been reported following transplantation with oral mucosal stem cell grafts. A second strategy would be to use cells of ocular origin, for example conjunctival epithelial cells. Among all stratified epithelial tissues, in fact, the conjunctival cells are the ones that more resemble the biological characteristics of corneal epithelial cells, thus suggesting that they may reprogram and serve some of the functions of corneal epithelial cells when transplanted onto the corneal surface. Recent studies have demonstrated the potential of human conjunctival epithelial cells cultivated onto amniotic membrane to be an alternative tissue source for replacement of the corneal epithelium, but results were limited to rabbit animal models of LSCD and observation period was relatively short (Tanioka et al., IOVS 2006;47:3820-27)(Ono et al., Mol Vis 2007;13:1138-43). Transplantation of autologous cultured conjunctival or oral mucosal epithelial stem cells would therefore make immunosuppressive therapy un-necessary and avoid the risks associated with allograft immunologic rejection. However, since these cells are not of cornea or limbus origin, it will be important to perform long-term studies evaluating the restoration of a clear and stable corneal epithelium free from opacities and new vessels.

 

Fondazione Banca degli Occhi del Veneto is currently pursuing this research topic. The aim is to evaluate whether new approaches based on transplantation of autologous stem cells from alternative sources (oral mucosa and conjunctiva) could be a potential therapeutic treatments for patients with bilateral LSCD, thus avoiding the need for immunosuppressive therapy and the risks and costs associated with allograft immunologic rejection.

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