Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral

Abstract

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. 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 transplanted 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 to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Dentin Mechanobiology: Bridging the Gap between Architecture and Function

“…While micropatterned platforms can mimic the architectural arrangements of dentin, dentinal cells are still confined to 2D physical contacts. Tooth organoids offer an alternative for replicating 3D spatial-temporal cell-cell interactions [ 151 , 152 , 153 ]. However, organoid development relies on self-assembly, resulting in random cell mixing and positioning within organoids without spatial arrangement between cells.…”

From Pluripotent Stem Cells to Organoids and Bioprinting: Recent Advances in Dental Epithelium and Ameloblast Models to Study Tooth Biology and Regeneration

“…Chorioallantoic membrane (CAM) assay ( in ovo ) : day 7 organoids were seeded with Matrigel onto the CAM of fertilized chicken eggs (9 days) for 1 week. Kim (2023) [ 89 ] Adult (9 week) mouse incisor organoids DMEM/F12 + 1X B27 , 1X N2 , N-acetylcysteine (1 mM), Y-27,632 (10 µM), 50% WNT3A conditioned medium, 5% RSPO1 conditioned medium, EGF (100 µg/mL), NOGGIN (200 µg/mL), FGF10 (100 µg/mL), dibenzazepine (10 mM), A83-01 (0.5 µM), nicotinamide (10 mM) day 0–14 : culture in expansion medium day 14–28 : suspension culture in expansion medium supplemented with SAG (concentration not provided by authors) Kidney transplantation : day 14 organoids were first cultured in suspension for 4 weeks and subsequently transplanted into the kidney capsule for 8 weeks. Underlined : Shared factor in all expansion media 1 SFDM: serum-free defined medium, see Hemeryck or Hermans et al for detailed composition [ 87 , 88 ]; 2 KSFM: keratinocyte serum-free medium …”

“…Recently, our group was the first to develop epithelial organoids starting from human and postnatal mouse tooth, followed by Kim et al who derived incisor organoids from adult mouse (Fig. 2 E; Table 4 ) [ 87 – 89 ]. Human tooth(-derived) organoids (TO) were established from the ERM obtained from the dental follicle of unerupted third molars [ 87 ].…”

“…Mouse TO were established from early-postnatal (day 7) human-resembling molars and ever-growing incisors, and from 9-week-old adult mouse incisor [ 88 , 89 ]. Both protocols rely on typical organoid medium components containing WNT pathway activation (WNT3A, RSPO1), BMP inhibition (NOGGIN), EGF (notably lacking from developed human TO medium), activin receptor-like kinase 5 (ALK5) inhibition (A83-01) as well as N-acetylcysteine, nicotinamide and fibroblast growth factor 10 (FGF10).…”

“…Importantly, both studies reported unique protocols able to mirror distinct aspects of AB-like differentiation in vitro [ 88 , 89 ]. Whereas suspension culture (which mirrors the loss of basement membrane between dental epithelium and dental mesenchyme during the transition from preAB to sAB) and SHH activation enable mirroring of the sAB phase (formation of crystals containing AMELX and AMBN), 3D culture in Matrigel with BMP and TGFβ activation predisposes toward the mAB stage (high ODAM/AMTN).…”