Burman and Sangwan rabbit limbal epithelium on human amniotic membrane (HAM) in vitro and then, after transplantation onto rabbit ocular surface, conﬁrmed the viability of the transplanted cultivated epithelium in vivo. Subsequently they showed that denuded HAM was better than intact cellular HAM for corneal epithelial cell culture (Koizumi et al 2000b). They adopted the culture system using epithelially denuded HAM with an underlying layer of lethally irradiated mouse 3T3 ﬁbroblasts. Their results showed that the expanded epithelium resembled a corneal phenotype with respect to the expression of K3 keratin (Koizumi et al 2001a, 2001b). Grueterich and colleagues (2002b) demonstrated that culturing limbal explants on an intact amniotic membrane without the use of a 3T3 feeder layer resulted in a limbal epithelial phenotype. Koizumi and colleagues (2002) demonstrated that both cell suspension and explant culture methods produced a healthy epithelial cell layer, with cells from the former being morphologically more superior. Currently most investigators prefer the explant culture technique (Tsai et al 2000; Grueterich et al 2002c; Koizumi et al 2000c, 2001b; Shimazaki et al 2002). The beneﬁts of using explants are that they are easy to prepare and there is no danger of damaging the corneal epithelium through enzyme treatment. The general principles of culturing the cells by explant culture technique involve the following steps: harvesting the limbal tissue (from the contralateral healthy eye in case of a unilateral LSCD, or from donor corneas for bilateral LSCD); selecting the appropriate carrier: a sheet of multi-layered epithelium, human amniotic membrane, collagen shields, or contact lens; preparation of human corneal epithelial medium; and explant cultures (Sangwan 2001). Conﬁrmation of growth can be done by various methods including-direct observation, whole mount stained prepara- tion, histopathology, immunohistochemistry, thymidine incorporation and by ﬂow cytometry using markers for cell cycle. Details of a few of the procedures can be found in an earlier publication (Vemuganti and Balasubramanian 2002). Although a number of investigators have included vari- ous reconstruction techniques through the use of autogenous conjunctiva, mucous membrane grafts, collagen lattices, synthetic implants, and cell-suspension cultures, the most widely accepted universal substratum for explant cultures is the HAM (Grueterich et al 2003). It was recognized and successfully reintroduced by Kim and Tseng (1995) for cor- neal surface reconstruction in rabbits. Amniotic membrane 494 is the innermost layer of the fetal or placental membrane and consists of an epithelial monolayer, a thick basement membrane, and an avascular stroma. Preserved HAM can be used a biological substrate without viable and active proliferative cells. It is thus nonimmunogenic and therefore does not require immunosupression when used as a graft for transplantation. Amniotic membrane serving as stem cell niche The role of the devitalized epithelium is not yet fully un- derstood. It has been shown that native, intact amniotic membrane (AM) epithelium contains higher levels of epi- dermal growth factor, keratinocyte growth factor, hepatocyte growth factor, and basic ﬁbroblast growth factor compared with epithelially denuded AM. However, Koizumi and col- leagues (2000b) indicated that denuded AM promotes better corneal epithelial cell colonization than intact AM does and that corneal cells from the limbal epithelium colonize denuded AM more readily than epithelial cells from the central cornea. Corneal cells colonize intact HAM much less quickly than denuded amnion. Moreover, migrating limbal SC on denuded HAM have a smooth, uniform lead- ing edge compared with the irregular raised edges of sheets grown on intact HAM. Morphologic observations further strengthen the support for epithelium grown on denuded HAM. The basal cells grown on bare amniotic membrane are nicely columnar, and the more superﬁcial cells seem fairly well differentiated into wing cells and surface cells. In contrast slowly growing epithelial cells on intact HAM do not take the appearance of normal corneal epithelium. However, expanded epithelium from limbal explants on intact AM adopts a limbal epithelial phenotype whereas that expanded on epithelially denuded HAM reveals a corneal epithelial phenotype (Grueterich et al 2002b). Wei and colleagues (2007) have further demonstrated that during ex vivo expansion on intact HAM some limbal epithelial progenitor cells migrate onto intact AM whereas some also invade the limbal stroma, very likely undergoing epithelial- mesenchymal transition. The HAM’s basement membrane contains type IV collagen, ln-1, Ln-5, ﬁbronectin, and col- lagen VII. The collagen IV subchain α2, is identical to that of the conjunctival BM. Ln play an important role in corneal epithelial cell adhesion. Overall the basement membrane facilitates migration of epithelial cells, reinforces adhesion of basal epithelial cells, promotes epithelial differentiation, and prevents epithelial apoptosis, that is, programmed cell death (Lee et al 2000). Clinical Ophthalmology 2008:2(3)