The large dendritic AQP cells
The large dendritic AQP1+ aldehyde dehydrogenase inhibitor formed an intricate network with N-cad+ limbal basal progenitor/stem cell clusters (Fig. 4). Melanocytes in the limbal area interact with limbal epithelial cells at a ratio of approximately 1:10, which is 3-folder higher than the average melanin unit of the skin (Hadley and Quevedo, 1966; Higa et al., 2005). Melanocytes were reported as one of the key components of the limbal stem cell niche (Hayashi et al., 2007; Higa et al., 2005). These observations suggest that the limbal stem cell niche consists of a unit of several different cell types closely associated with the limbal basal progenitor/stem cell clusters reported in this study.
Several studies have demonstrated that p63, a p53 isoform, is a useful marker for putative corneal epithelial stem cells (Di Iorio et al., 2005; Pellegrini et al., 2001), and p63 positive epithelial cell clusters were shown to have greater growth potential (Kawakita et al., 2009). The smallest cells with large N/C ratios are located in the basal limbal epithelium (Z. Chen et al., 2004; Romano et al., 2003), and K15 positive cells are also located in the basal limbal epithelium (Yoshida et al., 2006). In our previous paper, we also showed that N-cad plays an important role in the maintenance of the K15 positive cultivated limbal epithelial phenotype (Higa et al., 2009). Our cytospin data showed that N-cad+ cells were smaller, have a larger N/C ratio, and express both p63 and K15 (Fig. 3). Taken together, these data suggest that N-cad+ cell clusters shown in this study include the small putative corneal epithelial stem cells, if not the stem cells themselves.
One of the limitations of this study is that we were not able to clearly detect the expression of N-cad in the AQP1+ cells. This is probably due to the fact that the adhesion area between N-cad+ and AQP1+ cells is very small, probably mediated by stalk-like processes of approximately 1μm deduced from the observation by laser scanning confocal images and electron microscopy (Fig. 4E–H, Fig. 6B–D). In the bone marrow HSC niche, N-cad and β-catenin are asymmetrically localized between SNO cells and long-term HSCs (Zhang et al., 2003). These findings support the possibility that N-cad in the AQP1+ cells are only expressed at the tip of the cell attachment to N-cad+ limbal basal stem/progenitor cells.
Our study presents data demonstrating the requirement of N-cad in the interaction between N-cad+ limbal basal stem/progenitor cells and AQP1+ niche-like cells. We confirmed that N-cad+ cells were more adhesive to AQP1+ cells in calcium-containing media (Fig. 7), suggesting that this adhesion was probably calcium-dependent homotypic adhesion through N-cad. However, we observed a calcium independent cell adhesion between N-cad+ limbal basal stem/progenitor cells and AQP1+ niche-like cells as well. Therefore, we cannot rule out the possibility of adhesion molecules other than N-cad in the interaction between limbal stem/progenitor cells and AQP1+ niche-like cells.
Conclusions Although further studies are required, our findings indicate that large dendritic AQP1+ stromal niche-like cells exist beneath the N-cad+ limbal basal progenitor/stem cell clusters. Cell interaction may be direct adhesion with limbal basal progenitor/stem cells by Ca-dependent N-cad adhesion such as with NSO in the HSC niche.
Acknowledgments This study was supported by a grant of Advanced and Innovational Research program in Life Sciences from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K. H. The authors thank Ms. Kimie Kato and Ms. Junko Higuchi for their technical assistance, and the staff of the Cornea Center Eye Bank for administrative support.
Introduction Adherent, fibroblastic cell populations have been isolated from many connective tissues, and some have been found to contain a subset of local stem/progenitor cells. Some of these cell populations are suggested to be nonimmunogenic, and may have the ability to migrate to injured or inflamed sites. Those features support their potential clinical applications including treatment of immune, cardiovascular and degenerative diseases (osteoarthritis and osteoporosis), and their use as drug delivery vehicles (Caplan, 2007; Gimble et al., 2007).