Characterizing the precise molecular composition of the limb

Characterizing the precise molecular composition of the limbal niche has been another major limitation. It is known that components of BM vary according to location and this compositional heterogeneity may be responsible for differences in cell behavior (Kruegel and Miosge, 2010). On the ocular surface, the stromal matrix and BM of the corneal, limbal and conjunctival epithelia differ significantly (Schlozter-Schrehardt et al., 2007; Ljubimov et al., 1995; Tuori et al., 1996). Elegant tissue recombination studies using rabbit limbal and central corneal epithelial sheets placed on either limbal or corneal stroma have demonstrated that the limbal stroma maintained ‘stemness’, while the corneal stroma promoted differentiation, proliferation and apoptosis (Espana et al., 2003), further highlighting the critical nature of signals which emanate from stromal elements. Some of the more abundant proteins found in the limbal BM include collagen types VII, XVI (Adams et al., 2006), IV (Echevarria et al., 2011) laminin (LN)-α1, α2, β1 chains, agrin and vitronectin (VN) (Schlozter-Schrehardt et al., 2007; Ljubimov et al., 1995). Given these compositional differences, it is reasonable to propose that receptors for these ligands on adjacent epithelial gpr119 agonist are useful markers for identifying SC. A recent investigation from our laboratory identified VN as an abundantly expressed protein in the human limbal, but not corneal BM, which enhanced colony efficiency of limbal epithelial cells (Echevarria et al., 2011). Interestingly, studies involving human induced pluripotent SC and human embryonic SC validated the use of VN as an in vitro substrate for supporting pluripotency, without the need for fibroblast feeder cells (Braam et al., 2008; Rowland et al., 2010). VN is a glycoprotein that anchors to ECM molecules via its collagen or heparin binding domains (Schvartz et al., 1999), promoting cell adhesion and migration through Arginine–Glysine–Aspartate binding motifs (Ross, 2004) and is recognized by VN receptors including αvβ3 and αvβ5 integrins. Integrins are a superfamily of cell-surface receptors that comprise heterodimers of α and β subunits. Currently, 24 α and 9 β subunits have been identified, forming an array of proteins, which can also interact with growth factor receptors to transduce intracellular signals (Ross, 2004). Integrins may be ideal markers for SC as their expression on the plasma membrane provides a tethering site for isolating cells. The current investigation identified αvβ5 integrin as a candidate SC marker. Expression of this protein was used to enrich for limbal epithelial progenitor cells which formed holoclones in culture and expressed putative LESC markers. Global mRNA profiling of integrin expressing cells identified several interferon inducible genes whose function in the cornea is yet to be determined but may be involved in the migration and immunoregulation of LESC.
Materials and methods
Results
Discussion In this study we describe the localization of the VN receptor αvβ5-integrin on putative LESC (Figs. 1, 2, 7 and 8). Furthermore, cell-surface αvβ5 was used to tether, isolate and enrich a population of presumed limbal epithelial progenitor cells based on phenotypic and functional attributes (Figs. 5 and 6). Our recent description of the distribution of VN on the limbal but not corneal BM (Echevarria et al., 2011) is also corroborated in the current investigation (Figs. 1 and 2). Finally, after performing microarray analysis, the global transcriptional profile of these cells revealed enhanced expression of several previously undescribed immune-regulatory genes (Supplementary Table 2) that we propose play a key role in cell migration and maintaining LESC homeostasis through immune-protection. To improve on current enrichment and expansion strategies and to identify better markers of LESC, it will be necessary to determine the precise protein constituents of the niche. It is well documented that the BM composition of the cornea, limbus and conjunctiva differs considerably, with several factors more prominent in the limbal compared to the corneal and/or conjunctival BM (Schlozter-Schrehardt et al., 2007; Ljubimov et al., 1995; Tuori et al., 1996). VN is one such protein, which co-localized to vimentin+ clusters of basal limbal epithelial progenitor cells (Schlozter-Schrehardt et al., 2007). During embryological development, the ECM protein tenascin-C is widely expressed in preterm, less abundant in neonatal and restricted to the limbal border in the infant and adult human cornea (Maseruka et al., 2000). This pattern of expression resembles the distribution of LESC in the human fetal and adult cornea (Davies et al., 2009; Rodrigues et al., 1987) and suggests that deposition of ECM and BM proteins during development is critical for establishing the SC niche in adulthood. Our unpublished observations suggest VN may be locally synthesized by early progenitor cells during embryonic development and deposited on the BM prior to birth. It is also possible that VN is made by corneal mesenchymal cells or is blood-derived from the local limbal vasculature. Notably, fibronectin was widely distributed across the adult corneal, limbal and conjunctival BM (Supplementary Fig. 1) (Schlozter-Schrehardt et al., 2007), suggesting that it is less likely to act as a specific LESC support factor, although it has been shown to inhibit terminal differentiation of human epidermal keratinocytes (Adams and Watt, 1989).