Adult Dental Epithelial Stem Cell-Derived Organoids Deposit Hydroxylapatite Biomineral

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. Because enamel is acellular and has limited capacity to heal, regenerative technologies are highly desirable, yet have remained largely out of reach. This is in part due to the challenges of synthesizing crystallites with similar properties and assembling them in a mechanically robust structure. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When implanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model for the development of the dental epithelium and enamel, and an important first step towards regrowth or replacement of enamel. Organoids were grown from aDESCs embedded in Matrigel using media supplemented with Wnt3a, R-spondin1, and Egf (minimal); minimal + Noggin (+N); +N + Fgf10 (+NF); or +NF + dibenzazepine (+NFD) for 14 days.