It has been previously confirmed that SB selectively inhibit
It has been previously confirmed that SB-505124 selectively inhibits TGF-β and activin signaling more efficiently than its analog SB-431542 (DaCosta Byfield et al., 2004), which has been shown to induce neural differentiation when used in combination with Noggin (Chambers et al., 2009). IWP-2, on the other hand, functions as an inhibitor of the canonical Wnt pathway (Chen et al., 2009b) and was shown to induce cardiomyocyte differentiation when applied following a pretreatment with glycogen synthase kinase 3 inhibitor (Lian et al., 2013b). In our study, the initial induction of differentiation in the presence of these two factors, together with FGF, upregulated the expression of transcription factors active during early eye development, namely PAX6, PITX2, BMP4, and FOX1. Meanwhile, expression of the pluripotency markers OCT4, NANOG, SOX2, and c-MYC was downregulated. More importantly, the use of SB-505124, IWP-2, and bFGF during the induction stage of the protocol (SM condition) improved overall yields of p63-positive opioid receptor antagonist in each of the separate biological replicates when compared to induction without these factors (UM followed by maturation in CnT-30 medium). Expression of p63 peaked at the 30-day time point when up to 95% of the cells in SM condition were p63 positive and decreased to an average of 70% by the endpoint of the study (day 44). In the UM condition, the number of cells expressing p63 continued to increase to some extent after the 30-day time point, and by the endpoint of the study, approximately 60% of cells were p63 positive. In other words, corneal epithelial differentiation had progressed further in SM condition, marked by decreasing expression of p63 and increasing expression of CK3 and CK12, whereas differentiation in UM condition was less efficient in the given time frame. In contrast, differentiation under spontaneous conditions (in UM throughout the course of the study) was not sufficient for generation of p63-positive cells. Furthermore, even though there were no significant differences between conditions with regard to proliferation rates, because p63 was shown to be expressed in most cells that had undergone induction with SM, these p63-positive cells are likely the ones proliferating. In contrast, there seem to be other proliferating cell types present in the other culture conditions besides the p63-positive cells. Although an entirely specific marker for corneal epithelial progenitor cells is yet to be identified, transcription factor p63, especially its isoform ΔNp63α, has been linked to stemness and is highly expressed in the basal layers of the corneal epithelium and in the limbus (Kawasaki et al., 2006; Robertson et al., 2008). Mutations in p63 cause severe abnormalities, such as syndromes of ectodermal dysplasia affecting various ectodermal tissues, including the cornea (Shalom-Feuerstein et al., 2013), highlighting the importance of this transcription factor for proper corneal development. More importantly, p63 expression also appears to be clinically relevant, as it was discovered that cell cultures used for limbal stem cell transplantation containing more than 3% of p63-positive cells were associated with a 78% success rate, whereas transplants containing 3% or less p63-positive cells were successful in only 11% of patients with LSCD (Rama et al., 2010). To this respect, the high differentiation rate of p63-positive cells obtained with our protocol could be sufficient to gain good success rate in clinical applications. In our study, the adhesion to collagen-IV-coated substrate was significantly improved upon induction with SM. The ability to attach to collagen IV is considered to be important for epithelial cells, and several previously published differentiation studies have used this characteristic for selection of epithelial progenitor cells from limbal epithelial cell populations and, to a lesser extent, from differentiating pluripotent stem cells (Bian et al., 2010; Homma et al., 2004; Kumagai et al., 2010; Li et al., 2005). In addition, it has been shown that p63 is important for cell adhesion and migration in various epithelial cell lineages, including corneal epithelial cells (Carroll et al., 2006). Therefore, the high adhesion rate to collagen-IV-coated substrate observed in our study might be at least in part due to high expression of p63 after induction with SM.