Fig. 6: Differentiation of the aggregates into craniofacial mesenchyme-like cells.

a Scheme of the long-term culture and bright-field images of the aggregates. Aggregates were cultured with or without bFGF (basic fibroblast growth factor) from day 4. Scale bar, 400 µm. NCC: neural crest cell. Three independent cultures were used for experiment and representative images are shown. b Real-time PCR analysis of craniofacial mesenchyme-related genes. Data are presented as mean ± SEM (n = 3 independent experiments). Two-tailed Student’s t-test was used. Source data are provided as a Source Data file. c Schema of gene expression pattern in the first branchial arch. MN mandibular arch, MX maxillary arch. d Immunostaining of bFGF-treated aggregates on day 28 with antibodies for TWIST1 (green) and counterstaining with DAPI (blue). Scale bars, 200 µm. Three independent cultures were used for experiment and representative images are shown. e Immunostaining of bFGF-treated aggregates on day 28 with antibodies for DLX2 (green), LHX6 (red), and PRRX1 (blue). Scale bars, 100 µm. Three independent cultures were used for experiment and representative images are shown. f Immunostaining of bFGF-treated aggregates on day 28 with antibodies for BARX1 (green) and counterstaining with DAPI (blue). Scale bars, 100 µm. Three independent cultures were used for experiment and representative images are shown.