br Material methods br Results br Discussion Several

Material & methods
Results
Discussion Several LSC culture systems have been developed to re-create the LSC niche in vitro and to avoid the cross-contamination with non-human feeder molecular weight mg (Sharma et al., 2012; Xie et al., 2012; Chen et al., 2013; Mei et al., 2014). It has been suggested that feeder cells are required to maintain a stratified epithelial sheet and to regenerate a sufficient number of progenitor cells in culture (Miyashita et al., 2008). In the present study, LSCs were cultured with human BMSCs as feeder cells in different systems to determine whether these BMSCs were a suitable replacement of mouse 3T3 feeder cells. To compare the quality of different culture systems, we evaluated several parameters such as cell morphology, cell growth success rate, PD rate, the proportion of stem/progenitor cells, and the proportion of mature corneal epithelial cells. Based on the quantifiable measures that we evaluated, we determined that single LSCs could not be efficiently cultured on BMSCs in each culture method tested since differentiation in these cultures was increased (p63α data not shown). This finding is consistent with a previous observation (Omoto et al., 2009). LSCs cultured in the form of cell clusters achieve a higher expansion efficiency of the progenitor cell population (Kawakita et al., 2009; Gonzalez and Deng, 2013). In contrast, LSC clusters cultured in the 2D system produced cell outgrowths with a very heterogeneous morphology and a significantly low number of p63αbright cells; the presence of large differentiated cells at the center of the outgrowth, away from the feeder cells, suggested that nutrients secreted by the BMSC feeder cells could not reach this central area. In addition, the cell growth success rate was significantly lower in the 2D culture methods regardless of whether LSCs were seeded as single cells or as cell clusters. In the cultures of successful growth, the size of cell outgrowths was small and the cell morphology was consistent with that of differentiated epithelial cells at the center of the colony. These observations suggest that BMSCs can partially support the growth of LSCs in a 2D culture system. Apart from the insufficient nutrient supply, the 2D method has other disadvantages that need to be overcome if cells produced in this system are used in a clinical setting (Mei et al., 2014). First, both LSCs and feeder cells compete for the growth surface; LSCs push away the feeder cells as they grow, and this competition for the growth area might lead to a decrease in the number of feeder cells and an insufficient nutrient supply. Second, cross-contamination with the feeder cells is possible because both types of cells are grown in direct contact. To avoid these shortcomings in the 2D culture method, the 3D method was developed; this method has been shown to support the LSC phenotype and increase the expansion rate when mouse 3T3 feeder cells are used (Mei et al., 2014). LSCs grown in the 3D CC-BM system demonstrated the most homogeneous limbal epithelial morphology. The cultured epithelial cells were small and cuboidal in shape with very tight cell-to-cell junctions. The cell expansion rate and the percentage of K14+ and p63αbright cells in the 3D CC-BM system were equivalent to the control. Although p63α is expressed in both TA and stem cells, cells expressing high levels of p63α are usually located at the basal layer of the limbus where LSCs reside. Moreover, the only known predictor of clinical outcome is the percentage of the p63αbright cells (Rama et al., 2010). The proportion of K12+ cells in the 3D system was comparable to the control. The differences did not reach statistical significance and such low levels were clinically negligible. Our finding supports the hypothesis that the 3D culture system is better than the standard 2D system in mimicking the in vivo spatial environment of LSCs (Mei et al., 2014). In this 3D culture system, LSCs are evenly distributed and in close contact with BMSCs; this arrangement allows a more homogeneous niche support that is not present in the direct method of culture. Moreover, cross-contamination with feeder cells is avoided in this 3D method.