br Acknowledgments This work was supported by

Acknowledgments This work was supported by grants of the Basque Government (GIC/IT-589/13, SAIOTEK S-PE11UN124-125 and DKR-2012-59 to A. R.), the University of the Basque Country (UPV/EHU 243/2011 to I. G-B.), and the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No potential conflicts of interest were disclosed.
Introduction Limbal epithelial stem/progenitor CMX001 (LSCs) reside at the sclerocorneal junction (limbus) deep at the limbal epithelial crypt or lacuna that presumably constitutes the LSC niche (Dua et al., 2005; Shortt et al., 2007; Zarei-Ghanavati et al., 2011). Damage to the LSCs and their niche leads to limbal stem cell deficiency (LSCD). When LSCD is severe, normal homeostasis of corneal epithelial cells is impaired, and patients experience recurrent or persistent epithelial defect, corneal neovascularization, and scarring of the cornea that ultimately lead to functional blindness. Replenishing LSCs by transplantation of cultivated LSCs is an effective treatment to restore a normal cornel epithelial surface. The standard protocol to cultivate LSCs is to seed single LSCs directly on a monolayer of mouse 3T3 fibroblasts (Lindberg et al., 1993). The 3T3 cells may serve as surrogate niche cells secreting the necessary growth factors and cytokines that are important in the maintenance of LSCs. Efforts to culture LSCs have included the modification of the culture system to re-create the in vivo environment of the LSC niche and to avoid the use of animal components in the culture system (Sharma et al., 2012; Xie et al., 2012; Chen et al., 2013; Mei et al., 2014). This niche is thought to possess unique properties that provide a special microenvironment that regulates the LSCs (Daniels et al., 2006; Li et al., 2007). LSCs are in close proximity with their subjacent limbal stromal niche cells, which are presumed to be located mostly around and underneath the LSCs. Crosstalk among niche cells, extracellular matrix components, and soluble factors control the differentiation cues to replenish the cornea epithelium during normal homeostasis and upon injury. In some studies, LSC expansion efficiency was not optimal in a feeder-free system. Moreover, xenobiotic-free cultures appeared to have a lower clinical success rate than did the cultures using 3T3 feeder cells (Sangwan et al., 2011; Shortt et al., 2014; Zakaria et al., 2014); this reduction in clinical success could be due to the culture system not generating a sufficient number of LSCs. To maintain the LSC phenotype in vitro, the culture conditions would need to replicate the LSC microenvironment in vivo. Bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells to cultivate LSCs because BMSCs secrete factors required for epithelial cells proliferation, such as hepatocyte growth factor and keratinocyte growth factor (Omoto et al., 2009). However, the efficiency of LSC expansion using BMSCs has not been extensively evaluated. The proliferation rate and expression of markers that predict clinical outcome have not been measured (Rama et al., 2010). Our laboratory has developed a novel 3-dimensional (3D) culture system that has been shown to be an efficient, consistent, and reproducible culture system to expand LSCs (Mei et al., 2014). This 3D culture system allows LSCs to be in close proximity with feeder cells providing an even diffusion of growth factors and cytokines, and possible direct cell-to-cell contact between LSCs and feeder cells. The 3D system also addresses the following shortcomings of the traditional or standard 2-dimensional (2D) culture method: variation in nutrient diffusion results in nutrient gradients from the center of the LSC colonies to their periphery, and LSCs and feeder cells compete for the growth area in 2D systems.