Figure 7: Ameloblast and Odontoblast Co-culture Allows Further Maturation of ieAM into isAM.

(A) Schematic representation of the co-culture experiment between iAM organoids and DPSC organoids in suspension culture. The organoids were formed separately and then combined for 14 days in iAM base media. Immunofluorescence analysis revealed the expression of AMELX in iAM organoids (B) and the expression of CD146 (mesenchymal marker) and DSPP in DPSC/OB organoids (C). Alizarin red staining (D) indicated positive calcification in both organoid types, with DPSC/OB organoids showing more calcifications. (E) Schematic representation of the co-culture experiment between DPSCs as a monolayer and iAM embedded in Matrigel above it. Calcein, a fluorescent dye that binds to calcium, was added to the media containing a mixture of iAM base media and odontogenic media. (F) Three-dimensional reconstructed image from confocal images of the co-cultured organoids, showing association with Calcein and expression of ENAM at the center after 7 days (G). After 14 days, the organoids close to CD146-expressing DPSC/OB started to revert polarity towards DPSCs/OB while expressing AMELX (H), as depicted in the simplified diagram (I). (J) Schematic representation of the in vivo mouse experiment, involving the injection of day16-ieAM combined with DPSCs beneath the capsule of the right kidney in adult SCID mice. Kidneys were dissected and cryosectioned for further analysis. (K) Immunofluorescence staining showing ENAM-positive isAM in engrafted areas beneath the capsule. (L) Alizarin red staining indicating calcifications associated with isAM. (M) Immunofluorescence staining showing ENAM-positive isAM and high DSPP-positive DPSCs/OB (N). (O) Summary model proposing the interaction between ieAM and DPSCs/OB leading to the maturation of isAM.