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. 2017 Dec 21;10:426. doi: 10.3389/fnmol.2017.00426

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Copyright © 2017 Chen, Lu, Guo, Ni, Zhang, Zhao, Wu, Sun, Zhang, Tang, Li, Chai and Li.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Hedgehog signaling enhanced the hair cell (HC) differentiation of inner ear progenitor cells in vitro. (A) Diagram of the differentiation assay in Lgr5+ cell spheres. After allowing spheres to form for 5 days, the spheres were collected and cultured in medium that is suitable for cell differentiation for a further 7 days. The cells were stained with antibodies against Myo7a and with DAPI after 12 days of culture. (B) Spheres formed by the Lgr5+ progenitor cells generated some Myo7a+ cells when cultured in differentiation medium for 7 days in the control group. (C) The Shh protein-treated spheres generated more Myo7a+ cells. (D) The total number of Myo7a+ cells per well. (E) The average number of Myo7a+ cells per sphere (30–50 μm) in the control and Shh protein-treated groups. (F) Diagram of the direct differentiation assay. A total of 2000 sorted Lgr5-EGFP+ progenitor cells were cultured on 4-well plates at a density of 20 cells/μl for 7 days in serum-free medium. (G–I) Representative images of Myo7a immunofluorescence after 7 days of culture in the control, Shh protein-treated, and cylcopamine-treated groups. (J) The number of Myo7a+ cells increased significantly in the Shh protein-treated group and decreased significantly in the Hedgehog antagonist (cyclopamine, SANT-1, or vismodegib) group when compared with the control group. Data are presented as means ± SE. N = 5, *p < 0.05 vs. the control group. Scale bars (B–C,G–I) are 50 μm.