Fig. 2

Schema of deriving corneal cell phenotype from iPSCs. Human iPSCs treated with competitors of activin, and nodal pathways result in the inhibition of SMAD signaling inducing neuroectodermal progenitor (NEP) fate by activation of Zic and Fox gene family. Subsequent directed differentiation of NEPs to corneal epithelial cells (CEPs) having expression of Pax6, ABCG2, p63, and cytokeratin 12 and 13 is done by inhibiting TGFβ and WNT signaling pathways. To obtain CSKs, iPSCs are at first directed towards NCC phenotype by inhibiting TGFβ and BMP4 signaling using SB431542 and Noggin respectively. NCCs can be differentiated to keratocan and ABCB5-positive CSKs by following a co-culture system involving PA6 stromal cells for SDIA or by following a more defined culture method utilizing the bFGF and ascorbic acid (ascorpate-2-phosphate, A-2-P) signaling pathway. ZO-1 and Na,K-ATPase-positive CEnCs (see references [68, 78] for hCEnC markers) can be differentiated from NCC following a sequential differentiation procedure where the NCCs are first treated with a GSK3b inhibitor to activate the WNT/β-catenin pathway followed by treatment with SB431542 to inhibit TGFβ-mediated SMAD signaling. RA promotes terminal CEnC differentiation inhibiting while ROCK inhibitor promotes survival and enhances functional properties of the CEnCS [83, 84]