Efficient induction of functional ameloblasts from human keratinocyte stem cells

Abstract

BackgroundAlthough adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.MethodsCultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.ResultsTreatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.ConclusionsApplication of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human–mouse chimeric tooth germs.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0822-4) contains supplementary material, which is available to authorized users.

FGF8-mediated signaling regulates tooth developmental pace during odontogenesis

“…Our previous studies have showed that epithelial sheets of human keratinocyte stem cells, when recombined with E13.5 mouse dental mesenchyme, can be induced to differentiate into enamel-secreting ameloblasts in only 10 days and apoptotically degraded in 4 weeks to achieve small-sized chimeric teeth with similar size to mouse ones, whereas the mouse secondary branchial arch epithelium, when recombined with the human dental mesenchyme from the bell-stage, remains in an enamel-secreting status after 15-week ex vivo culture and acquired large-sized teeth in the end, a size similar to human deciduous teeth (30, 38, 39) . These results strongly imply that the duration of epithelial differentiation and size control in tooth development could be dominated by the mesenchymal component in humans and mice.…”

“…A heterogenous recombination study carried out between the mouse and rat molar tooth germs has suggested that tooth size is determined by the dental mesenchyme (Cai et al, 2007, Jiang et al, 1999. Additionally, our recent studies showed that, in comparison with natural developmental pace, the differentiation duration of the mouse dental epithelium or non-dental epithelium into ameloblasts when induced by the human dental mesenchyme is significantly extended, whereas that of the human dental epithelium or epithelial stem cells into ameloblasts when induced by the mouse dental mesenchyme is significantly shortened (Wang et al, 2010, Hu et al, 2018. These results are consistent with the difference of development progression speed between human and mouse teeth (Zhang et al, 2005), suggesting a dominant role of the dental mesenchyme in regulation of tooth developmental tempo.…”

Hard Dental Tissues Regeneration—Approaches and Challenges

“…The generation of a whole tooth was accomplished by several methods, such as “organ germ” or “bioengineered organ germ” methods [ 200 ], stimulation of the formation of a new tooth (or third dentition) [ 201 ], engineering scaffolds of dental tissues [ 202 ], gene-handled tooth regeneration [ 203 ], chimeric tooth tissue engineering [ 111 , 204 ], in situ tooth regeneration by stimulating the tooth replacement ability [ 198 ] and cell–cell or tissue–cell recombination via embryonic tooth germ cells [ 200 , 205 , 206 , 207 , 208 , 209 ]. Currently, the main research directions for whole tooth regeneration are focusing on the in situ tooth regeneration by stimulating the tooth replacement ability, bioengineered organ germ, and tissue engineering approaches [ 30 , 197 , 198 ].…”

Tooth Formation: Are the Hardest Tissues of Human Body Hard to Regenerate?

“…However, unlike other mineralized tissues of the human body, enamel cannot be regenerated due to its acellular nature. Although several cell sources were shown to have amelogenic capacity including keratinocyte stem cells, epithelial cell rests of Malassez (ERM) from periodontal ligament, odontogenic oral epithelial stem cells (OEpSCs), adipose tissue-derived mesenchymal stem cells (AT-MSCs), and iPSCs [87][88][89][90][91][92].…”

“…Studies in dental organotypic cultures and transgenic mice also point out the importance of the mentioned pathways in dentogenesis. For example, Shh in combination with FGF8 was recognized as a stemness promoting ligans for ameloblast precursors (human skin fibroblasts) in a human-mouse chimeric tooth [87], while Runx2 was shown to have an affinity for the amelotin promoter and regulates its expression during the enamel maturation stage [95]. Regarding amelogenin turnover, a novel role of cytoplasmatic B-cell CLL/lymphoma 9 protein (Bcl9), its paralog B-cell lymphoma 9-like protein (Blc9l) and interaction partners Pygopus 1/2 (Pygo1/2) is proposed to play a role in amelogenin secretion [96].…”