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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Cell Rep. 2013 Nov 27;5(5):10.1016/j.celrep.2013.10.048. doi: 10.1016/j.celrep.2013.10.048

Figure 5. Identification of putative pre-placode and directed differentiation towards human lens placode lineage (see also Figure S6).

Figure 5

A) Immunocytochemical analysis for TFAP2A and PAX6 at day 3 of differentiation. B) Time course analysis at day 5, 7, 9 and 11 of PIP differentiation show co-expression of TFAP2A and PAX6 at days 7 and 9 of differentiation. C) Time course analysis at day 5, 7, 9 and 11 of N-SB differentiation shows lack of TFAP2A expression but expression of PAX6 in CNS neuroectodermal cells. Scale bars in A–C correspond to 25 μm. D) Temporal analysis of PAX3 gene expression during PIP versus NSB protocol. E) PAX3 expression levels following treatment (days 7–11) with activators or inhibitor of FGF (FGF8, SU5402) and WNT signaling (CHIR99021, XAV939) during PIP. F) PAX6 expression levels using same treatment as in (E). F) Results of a four signaling pathway screen (modulators of BMP, FGF, WNT and SHH signaling added at days 7–11 of PIP). Induction of the lens placode marker PITX3 by qRT-PCR was observed upon treatment with activators of BMP signaling (BMP4) or inhibitors of FGF signaling (SU5402). G) Modified placode cultures differentiating into Crystalline+ cells with mature lens fiber morphologies by day 57 of differentiation. Scale bars in (G) correspond to 50 μm. Error bar represents SD. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 compared with control PIP condition (n = 3 independent experiments).

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