Tooth-like structures formed from H1-ESC line and hiPSC lines in 3 weeks. (A) Left two columns: tooth-like structures after and before isolated from the surrounding tissues from H1-ESC and ifhU1-iPSC groups (tooth is outlined by a dotted line); right three columns: HE and immunohistochemical staining (IHC) of relative tooth sections (H1-ESC: sagittal section; ifhU1-iPSC: cross section), showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es), and a layer of ameloblasts (Am, high magnification in the blue box) in both groups. Positive Amel (black arrows) expresses in these ameloblasts with the papillary layer ( black arrowheads at high magnification in the green box). Scale bar: the upper and bottom figures in the same column share the same scale bar as 500, 500, 400, 100, 100 μm from left to right orderly. (B) IHC (top, HLA-I in cytoplasm) and IF (bottom, hNA in nucleus) staining in cross sections of the ifhU1-iPSCs derived tooth: human iPSC derived cells and tissues including ameloblast layer (arrowheads), cyst epithelial cells (arrows) and epithelium-derived cyst (*). DAPI is shown in blue. Scale bars: 200 μm. (C) Image of a piece of bone detected from E14.5 mouse dental mesenchyme being transplanted under kidney capsule for 3 weeks with positive BSP expression. Scale bars: 1000, 100, 100 μm orderly. (D) Nano-indentation analyses, Left: representative images of fractured enamel and dentin surfaces in adult human tooth group (human), group of 3-week mouse tooth from E14.5 tooth germs (Mouse-WT), regenerative tooth groups from H1-ESCs and ifhU-iPSCs; right: hardness and elastic modulus of enamel and dentin in above groups (each group: n=3). (E) Raman spectroscopy analyses of enamel and dentin from human, mouse, hESCs, and ifhU-iPSCs showed great similarity for all groups on Raman peaks. (F) Efficiencies of tooth-like structures for H1-ESC line and 8 iPSC lines.