Fgf Signaling Inhibition Promotes The Generation Of Non-Neural Ectoderm

Generating non-neural ectoderm (NNE) constitutes a vital procedure for constructing tooth organoids from human induced pluripotent stem cells (hiPSCs). While multiple protocols exist for inducing NNE, the optimal induction conditions remain elusive, thereby hindering the efficient production of NNE. hiPSCs were cultured in monolayer. The medium was supplemented with various concentrations of small molecules and cytokines to modulate the sinaling pathways involved in the development of NNE, including SU5402 (an FGF signaling inhibitor), retinoic acid (RA), BMP4, SB431542 (a TGF-β signaling inhibitor), and LDN193189 (a BMP signaling inhibitor), either individually or in combination. The expression of NNE markers (DLX3, DLX5, and TFAP2A) and markers of other germ layers was examined by qRT-PCR and immunostaining to evaluate the generation of NNE fate. The individual addition of 1 μM RA, 1 μM SB431542, or 10 μM SU5402 significantly upregulated the gene expression of DLX3, DLX5, and TFAP2A. Combinatorial treatments indicated that supplementing RA with BMP4 did not enhance the expression of these markers, whereas the addition of BMP4 to SB431542 increased DLX3, DLX5, and TFAP2A expression within a specific concentration range of BMP4. Among the RA, SB431542 + BMP4, and SU5402 groups, the SU5402 treatment most potently induced both gene and protein expression of DLX3, DLX5, and TFAP2A. Notably, pre-treatment with LDN193189 and SB431542 in SU5402 group did not suppress the expression of markers associated with other germ layers. Inhibition of the FGF signaling pathway by SU5402 promotes NNE generation more effectively than other established protocols.