Delhi Journal of Ophthalmology

Limbal Epithelial Stem Cells in Corneal Regeneration

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#  Shweta Sharma, *M.Vanathi, † Sujata Mohanty, *Radhika Tandon
# Sanford Burnham Medical Research Institute, La Jolla, California, USA
* Cornea and Ocular Surface Services, 
Dr Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, New Delhi, India,
† Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India

Corresponding Author:

Radhika Tandon
Professor of Ophthalmology, 
Cornea & Ocular Surface Services
Dr R.P Centre for Ophthalmic Sciences, 
AIIMS, New Delhi, India
E-mail: radhika_tan@yahoo.com

Published Online: 25-SEP-2013

DOI:http://dx.doi.org/10.7869/djo.2013.9

Abstract
The cornea is the transparent and outer most part of the eye, which is responsible for clear vision. The homeostasis of corneal epithelium is maintained by stem cells (SCs) located at the limbus, which is a transitional zone between cornea and conjunctiva. Due to some conditions limbal stem cells (LSCs) can be destroyed or lost and this can cause limbal stem cell deficiency (LSCD). Conventional non-surgical management and corneal transplants are not enough to treat LSCD. Recent advances in tissue engineering have made possible to rejuvenate the LSC deficit ocular surface with ex-vivo cultivated limbal epithelium and this is possible to achieve from a significantly small limbal biopsy.

In spite of all progress made in this field, it is still questionable to identify LSCs based on the biomarkers. This review article will focus on the biology of LSCs and their emerging trend in the field of regenerative medicine. Apart from that this article will also highlight different culture techniques to cultivate LSCs and novel biofunctional scaffolds.

Keywords :

Advancements in the field of tissue engineering have made it possible to replace diseased or dysfunctional tissue with custom fabricate tissue. In the cornea, the use of lineage committed stem cells have become the popular tread to alleviate corneal stem cell deficiency. This review will discuss about the stem cells of corneal epithelium, limbal stem cell biology, and their use as an emerging trend in the field of regenerative medicine.

Stem Cells of Corneal Epithelium

The human ocular surface is a complex biological structure, which is responsible for protection of the cornea and also for maintaining its clarity. It is covered with the highly specialized epithelium such as corneal, conjunctival and limbal. The corneal epithelium can be markedly distinguished from the neighboring conjunctival epithelial cells by a transition zone termed as “limbus”. The conjunctival epithelium is occupied by goblet cells[1], which are unicellular mucin-secreting glands and are considered to be primary source of the mucin of the tear film.[2,3] Goblet cells play a very vital role in maintaining the integrity of the ocular surface, as mucin deficiency has been a concern in several ocular surface diseases. Adult cornea is an avascular tissue composed of mainly three specialized layers (Figure 1).



(A) Corneal Epithelium
This is a non-keratinised stratified squamous epithelium, which makes approximately 10% of the total corneal thickness. It is usually 5–7 layers thick and is further divided into three layers. The outer most layer is differentiated squamous cells with numerous microvilli on the apical surface. By increasing cell surface area these microvilli provide close association with the tear film.

Squamous layer acts as a protective barrier by forming highly resistant tight junctions between cells hence prevent the entry of detrimental agents into the intraocular space.[4] Secondly, the underlying suprabasal cells or wing cells, which rarely undergo division. These cells migrate superficially to differentiate into squamous cells.

The inner most basal cells consist of a single layer of columnar cells with several important functions including generation of new suprabasal cells, secretion of matrix factors critical for basement membrane and stromal function and helps in organization of hemidesmonsomes and focal complexes to maintain attachment to the underlying basement membrane. These functions are suggested to be important in mediating cell migration in response to epithelial injury.[5]

 (B) Corneal stroma

Stroma is separated from epithelium by Bowman’s layer. It makes around 90% of the entire thickness of the cornea. It’s made up of collagens (types I, V, and VI) and proteoglycans (decorin, associated with dermatan sulphate, and lumican, associated with keratan sulphate). Keratocytes are randomly scattered in the collagen layer of stroma. Collagen fibrils are mainly responsible for corneal transparency.[6]
 
(C) Corneal endothelium

Descemet’s membrane (DM) separates endothelium from stroma. This is made up of a single layer of cells. They are mainly involved in pumping out water from the corneal stroma and allow corneal transparency. Corneal endothelial cells do not proliferate in-vivo unlike the epithelial cells.

The transitional zone between the cornea and the bulbar conjunctiva is referred to as the limbus.[1,7] Limbus rim is around 0.5mm wide horizontally and 2mm vertically. The limbus harbors the stem cells of the corneal epithelium[8,9] in a very protective environment. Limbal epithelium consists of several organized layers, devoid of goblet cells and populated by Langerhans cells and melanocytes (Figure 2). The melanocytes are supposed to guard the limbal stem cells (LSCs) from harmful ultraviolet rays.[10] They also quench UV-induced oxidant formation in the cornea epithelium by their anti-oxidative activity.
 
The deep projections into vascularized limbal epithelium are called “palisades of Vogts”.[11] They not only provide nourishment to the LSCs[9] but also protect them from shearing external forces.12 Recently Dua and Shanmuganathan (2005) have identified some projection like structures in palisades of Vogts and named them as ‘limbal epithelial crypt’.13 These crypts, which house putative LSCs predominantly, occur on the superior and inferior cornea where they are normally covered by the eye lids.[14]



LSCs characteristically possess several unique, inherent properties, as described below.[15]
  1. The cytoplasm of LSCs appears primitive in nature and contains few differentiated products. They possess a large nuclear to cytoplasm content (high N/C ratio).
  2. LSCs have a high capacity for self-renewal and potential of error-free cell division. Error-free proliferation is essential, as any genetic error at the level of stem cells will pass on to the whole progeny of cells, resulting in abnormal cellular genotype and phenotype.
  3. Stem cells have a long life span, which might be equivalent to the life of the organism in which they reside.
  4. Stem cells exhibit extremely low rates of proliferation (indicates low mitotic activity). They have a long cell cycle time or slow cycling.
  5. LSC can undergo symmetric or asymmetric division depending upon the conditions. When cell undergoes asymmetric division, one of the daughter cells remains as its parent and serves to replenish the stem cell pool, whereas the other daughter cell is destined to divide and differentiate into specific tissue type cell. On the other hand, the asymmetric division may be determined by the local environment, which induces otherwise similar daughter cells to behave differently. 

The Destruction or Dysfunction of Stem Cell Niche

The healthy and transparent cornea is required for clear vision, which in turn ismaintained by LSC population. The destruction of LSC or their niche can cause Limbal Stem Cell Deficiency (LSCD). The LSCD can be induced by two factors namely acquired or congenital (Table 1).[16,17] The condition can result in chronic ocular discomfort, photophobia, pain, compromised visual acuity or blindness. Destruction of limbus barrier results in the invasion of phenotypically different conjunctival epithelium and its associated blood vessels over the corneal surface. This phenomenon is called corneal conjunctivalization, which is the clinical hallmark of LSCD and as a result patient loses corneal clarity and vision. Conjunctivalization can be detected by presence of goblet cells on impression cytology (Figure3).





Ocular Surface Reconstruction by Limbal Stem Cell Transplantation

The stem cell deficiency could be partial or total depending upon the extent of limbus involvement with the underlying disease process. Various strategies are followed for managing LSCD and the treatment can be tailored depending at its extent (Flow Chart 1). If the damage is partial then continuous application of topical lubricants; anti-inflammatory agents and the use of autologous serum drops can heal the epithelial defects.[18-21]



In more advanced stage of partial LSCD, especially where only central cornea is affected surgical intervention is required. The abnormal corneal epithelium can be removed and amniotic membrane transplantation can be done.[22] This allows the denuded cornea, to resurface with cells derived from the remaining intact limbal epithelium. Total or severe LSCD can be treated by grafting viable limbal tissue obtained from the healthy donor eye. This procedure may help in replenishing the stem cell pool and can restore the damaged corneal surface.[23,24] Variety of procedures such as cadaveric keratolimbal allograft (KLAL), live related conjunctival limbal allograft (Ir-CLAL) and limbal autograft are available for Limbal stem cell transplantation (LSCT).[6]

Advances in tissue engineering techniques now provide an alternative to overcome the limitation of limbal tissue available for transplantation. Pellegrini et al, 1997 first showed that the corneal progenitor cells located in the limbus can be cultured to generate cohesive sheets of authentic corneal epithelium, and that cultured corneal epithelium can effectively restore the diseased corneal surface.[25] Various published reports have elaborated the results of cultured LSCT.[26-32] However, case selection and methods used for diagnostic criteria are variable. Moreover, in developing countries much remains to be done; to study as to how best these state of the art techniques can be adapted to meet the local needs. The overall success rate in various cultured LSCT studies is approximately 70 – 75% based on transparency, integrity, and stability of the corneal epithelium, which is also concur with our clinical results.[33] In this study[33], we reported the success of ex-vivo cultivated limbal epithelial cell sheet transplantation using simple and cost effective approach. The primary objective of this study was to refurbish a damaged ocular surface by transplanting cultivated limbal epithelial cells to restore the corneal surface and to reinstate the limbus function. The results demonstrated the safety and efficacy of LSCT procedure in our setup with the clinical success rate of 74% (82% in autografts and 56% in allografts). The successful restoration of damaged ocular surface may be due to the regeneration of demolished LESC niche, which may in turn repopulate the stem cells crucial for maintaining the epithelial integrity. In one of our report[34], we further described our experience using a meticulous approach to reconstruct the severely damaged ocular surface by combining our previously reported LSCT method with the deep anterior lamellar corneal transplantation using donor corneas to restore corneal clarity and vision by reducing corneal vascularisation. Further more histopathology of excised host lamellar corneal buttons demonstrated organised corneal epithelial morphology with the expression of CK3/12 in rejuvenated corneal.[34]

The technique of ex-vivo expansion of LSCs was based upon the pioneer method developed by Rheinwald and Green[35] for the cultivation of epidermal keratinocytes. Generally, there are two approaches being used for cultivation of limbal epithelial cells, namely the explant culture system and the suspension culture system. In explant culture, Human amniotic membrane, (HAM) is often used as both a substrate and a carrier. Limbal biopsy tissue is allowed to adhere to the amnion prior to being submerged in culture medium, which stimulates the limbal epithelial cells to migrate out of the biopsy and proliferate on the amnion.[27] Once confluence is reached, the multi-layered epithelium sheet can be used for transplantation (Figure 4). The suspension culture system involves separation of limbal epithelial by using dispase and trypsin prior to seeding either onto amniotic membrane or onto growth-arrested 3T3 fibroblasts (feeder layer).[36] After two weeks, the epithelial sheet is transferred to the ocular surface by using a contact lens, collagen shield or fibrin gel. When the suspension of single limbal epithelial cells is seeded onto amniotic membrane, they are usually co-cultured with a layer of growth-arrested 3T3 fibroblasts in the bottom of the dish.



It is reported that preparation of the HAM may influence the phenotype of the cultured limbal epithelial cells. Grueterich et al[37] have demonstrated that the culture of LESCs on amniotic membrane with an intact amniotic epithelium may result in a more stem-cell-like phenotype than de-epithelialised amnion. HAM is currently the most commonly used substrate for LESCs cultivation and transplantation.[38-41] Although the results are quite promising, amniotic membrane does have some shortcomings. One of the major issues is ensuring the biosafety of HAM in disease transmission, e.g., HIV, hepatitis B and C as well as from bacteria and fungus which will grow readily on HAM. Thus, procuring and storing HAM is a serious concern. In addition, as a natural product, consistency of HAM cannot be controlled. From a surgical standpoint, the physical structure of HAM does not provide significant mechanical strength to act as a tectonic base for support of the sclera or cornea. HAM also has an inherent semi-opaque nature, which impedes post-operative visual acuity until the tissue is remodeled (which can occur over a period of days to months).[42,43]

Advances in Ocular Surface Tissue Engineering

Nanotechnology has the potential to solve above mentioned problems by fabricating desired biocompatible materials to construct a functional tissue engineered ocular surface. This technology is based on the same principle as performed with HAM as a substrate. Limbal biopsy can be harvested and grown on an appropriate matrix or scaffold and subsequently transplanted to diseased eye. Various extracellular matrices have been used previously for limbal epithelial stem cell expansion such as fibrin, collagen scaffold, temperature responsive cell culture surfaces, human anterior capsule, natural and synthetic scaffolds etc.[44-53] Limbal epithelial cells expanded onto a fibrin substrate showed promising results; the corneal surface was covered by a transparent normal-looking epithelium and their visual acuity had also improved.[44] Nishida et al, (2004) have developed a temperature-sensitive sol-gel transition for the transfer of intact epithelial sheets for corneal resurfacing. While an excellent approach for avoiding the damage caused by trypsin in detaching cells, this does not solve other problems that need to be addressed in corneal transplantation. In this system, the temperature sensitive surface was deposited onto a culture dish and then oral mucosal epithelial cells were grown as a sheet of cells on this dish as an alternative source of autologous epithelium for corneal transplantation. They are then detached by changing the temperature and the sheet of cells was grafted onto the rabbit eye.46 In one of our studies[54], we have proposed Poly-e-caprolactone (PCL), which is a synthetic aliphatic polyesters bioresorbable and biocompatible as an excellent and biocompatible scaffold for LSC expansion (Figure 5). 



In previous years extensive research had been conducted on PCL biocompatibility and efficacy to get it approved by the U.S. Food and Drug Administration (FDA) for a number of medical and drug delivery devices.[53-55] In ophthalmic application, PCL has already been explored as a carrier[53] due to its in-vivo biocompatibility as it does not induce any immunological reactions after degradation. Our preliminary studies have shown that the PCL polymer provides a suitable alternative for overcoming the shortcomings of natural and synthetic polymers, resulting in a new biomaterial with good biocompatibility and improved mechanical, physical and chemical properties (Figure 6).



Apart from these advancements one of the most imperative and intricate challenge is in the field of LSC biology is their identification. Many groups are working on LSCs and recently many developments have been made into the clinics but their biology is still poorly understood primarily due to lack of knowledge about the nature of LSC. The literature reflects many attempts to identify LSC using a specific marker but yet no single, reliable marker has been found. Based on the previous reports a number of markers for LSC have been proposed, which are summarized in (Table 2).[56-70]



Conclusion

LSCT is widely used as a common technique for reconstructing damaged ocular surface based on its promising clinical results. Therefore, it may be reasonable to improve the culture environment following xeno–free conditions under GMP and GLP guidelines. It may be also wise to explore the alternative autologous cell sources, such as oral, nasal, and even hair follicle stem cells, which will replace need of allogeneic cells and the associated long –term immuno-suppression.

Financial & competing interest disclosure

The authors do not have any competing interests in any product / procedure mentioned in this study. The authors do not have any financial interests in any product / procedure mentioned in this study

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