nucleoside transporters br Experimental Procedures br Author

Experimental Procedures
Author Contributions
Acknowledgments
Introduction The cornea is a multilayered, transparent, and avascular structure forming the anterior part of the eye. Its outermost layer, the corneal epithelium, is exposed to the external environment and thereby needs to be rapidly regenerating and stratified. It is renewed by limbal stem cells, a type of tissue-specific stem cell located in specialized niche areas in the corneoscleral junction called limbus (Echevarria and Di Girolamo, 2011). Diseases affecting the cornea are a major cause of blindness worldwide and one of the leading causes of vision loss after cataract, with nearly 70% of corneal blindness being due to limbal stem cell deficiency (LSCD)—a disease characterized by abnormal corneal epithelial maintenance, resulting in conjunctivalization of the corneal surface (Ahmad, 2012). LSCD may be caused by acute trauma, such as chemical or thermal injury, or various chronic or genetic conditions (Notara et al., 2010; Osei-Bempong et al., 2013). Several different surgical techniques have been implemented to treat LSCD. One approach is to use cultivated limbal epithelial transplantation (CLET). However, this method is only possible if enough healthy limbal tissue is available, and long-term results show a considerable amount of variation in success rates. This is especially true in case of allogeneic transplantation, which also requires the use of long-term systemic immunosuppression (Baylis et al., 2011). In search of novel therapies for corneal disorders, alternative cell sources have been investigated, including hair-follicle stem cells, mesenchymal stem cells, and umbilical-cord-lining stem nucleoside transporters (Blazejewska et al., 2009; Reinshagen et al., 2011; Reza et al., 2011). One of the techniques enabling the use of autologous cells, cultivated oral mucosal epithelial transplantation (COMET), has been extensively studied, giving promising results for stabilization of the ocular surface. Generally, the main problems with COMET, as with CLET, include variation in success rates, use of serum and animal-derived materials in the culture protocols, and peripheral corneal neovascularization (Chen et al., 2009a, 2012; Hirayama et al., 2012; Kolli et al., 2010; Nishida et al., 2004; Satake et al., 2011; Sotozono et al., 2013). Thus, it is important to further develop functional cell-based modes of treatment for corneal defects. Human pluripotent stem cells (hPSCs) have a wider differentiation potential than tissue-specific stem cells, providing an unlimited source of cells. Human induced pluripotent stem cells (hiPSCs) in particular provide exciting new possibilities in the field of personalized medicine and disease modeling (Takahashi et al., 2007). The first study to successfully differentiate corneal epithelial-like cells from hPSCs used medium conditioned by limbal fibroblasts as a way of replicating the corneal stem cell niche (Ahmad et al., 2007). Since then, a few other studies have been published, all relying on various undefined or animal-derived components, such as feeder cells, amniotic membrane, or conditioned medium, alone or in combinations (Hanson et al., 2013; Hayashi et al., 2012; Hewitt et al., 2009; Shalom-Feuerstein et al., 2012). Using defined differentiation conditions free from animal-derived products and serum would diminish batch-to-batch variation, thereby minimizing the potential risk of animal pathogen transmission, immune reactions, and graft rejection (Kaur et al., 2013; Martin et al., 2005). Consequently, the repeatability and consistency of differentiation, as well as the safe use of the resulting cell populations in patients, would improve.
Results
Discussion In this study, we chose to examine the effects of a combination of two small-molecule inhibitors, SB-505124 and IWP-2, together with FGF, on differentiation of hiPSCs toward eye precursors and further toward corneal epithelial cells. During embryogenesis, corneal epithelium originates from the head/ocular surface ectoderm (Collomb et al., 2013; Wolosin et al., 2004). Although many of the developmental mechanisms and signaling routes remain elusive, it is known that blocking TGF-β/Nodal and Wnt/β-catenin-signaling pathways is required for head/ocular surface ectoderm development (Arkell and Tam, 2012; Dupont et al., 2005; Fuhrmann, 2008; Gage et al., 2008). Ectodermal placodes play a major part in the development of vertebrate cranial sensory organs and have been shown to emerge from a common preplacodal region (Streit, 2007). Preplacode induction in the naive ectoderm is promoted by a combination of FGF expression together with Wnt and bone morphogenetic protein (BMP) antagonists (Litsiou et al., 2005). Replication of these developmental cues by first blocking BMP signaling with Noggin to induce ectoderm formation, followed by activation of FGF signaling, was shown to initiate lens-progenitor cell differentiation from hESCs under chemically defined culture conditions (Yang et al., 2010). In a recently published study, a small-molecule inhibitor of the Src family kinases was shown to promote simple epithelial differentiation of hPSCs by downregulating canonical Wnt signaling (Lian et al., 2013a). Moreover, there is recent evidence showing that small-molecule inhibition of the TGF-β- signaling pathway improves ectodermal differentiation of hiPSCs (Shalom-Feuerstein et al., 2013).