Fig 1: Neural crest-like cells derived from induced pluripotent stem cells (iPSCs) (iNCLCs) contribute to vascularized dentin–pulp complex regeneration in vivo. (A) iPSCs formed neural spheres under suspension culture. (B) The spheres attached to fibronectin-coated dish, and the cells migrated out from the spheres. (C) The morphology of the migrated cells after one time passage. (D,E) Reverse transcription PCR (RT-PCR) and immunocytochemistry were performed to evaluate the expression of neural crest-specific markers including p75 and AP2-a in iNCLCs. (F) Multilineage differentiation of iNCLCs. Osteogenic differentiation, positive staining with Alizarin Red S; adipogenic differentiation, positive staining with Oil Red O; chondrogenic differentiation, positive staining with Safranin O. (G) HE staining images of each group (the right two panels are the magnification of the boxed region; black arrowheads indicate odontoblast-like cells, yellow dash line area indicates newly formed dentin, and green arrowheads indicate blood vessel). (H) Histomorphometric analysis of newly formed vessel area; (I) Dentin sialophosphoprotein (DSPP) immunohistochemistry of each group (the right two panels are the magnification of the boxed region; dash blue line area indicates newly formed dentin); mouse femur bone tissue was treated as negative control group. (J) CD31 immunohistochemistry of each group (green arrowheads indicate blood vessels). (K) Transplanted tooth scaffold/iNCLCs complex formed teratoma, containing three germ-derived tissues including keratin-containing epidermal tissues (ectoderm), cartilage (mesoderm), and gut-like epithelial tissues (endoderm). *P < 0.05.