Fig. 1

An overview of differentiation of mESCs to SG placodes. A Schematic representation of the early development of mouse SMG. B Culture protocol and schematic illustration of in vitro generation of SG placodes from embryonic stem cells (ESCs). NNE: non-neutral ectoderm; OE: oral ectoderm, SMG: submandibular glands. C BMP4 promoted the differentiation of oral ectoderm from mESCs. BMP4 increased the expression of Pitx1 in a dose-dependent manner. The mRNA expression was qualified by qPCR. The Ct values were compared to aggregates treatment without BMP4 (control: CTRL). D Immunofluorescence staining showed that Pitx1 positive oral ectoderm in the outer layer of aggregates at day 4. Scale bars, 100 μm. E Phase-contrast bright-light images represented the morphology of PSC-derived SG placodes during differentiation. Scale bars, 200 μm. F As differentiation proceeds, qPCR showed the increased expression of salivary gland progenitor markers. The Ct values were compared to aggregates on day 1. All qPCR results are presented as the fold change compared with the mean ± S.D. and were normalized to GAPDH in three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired, two-tailed Student’s t-test