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. 2023 Apr 20;18(5):1166–1181. doi: 10.1016/j.stemcr.2023.03.011

Figure 1.

Figure 1

Development and characterization of organoids from mouse molar and incisor

(A) Schematic of experimental setup for derivation of TOs from early-postnatal (PD7) mouse molar and incisor.

(B) Progressive development (bright-field pictures) of TOs after initial seeding (passage 0 (P0) day 1 (d1)), 7 days after initial seeding (P0 d7), and after long-term culture for more than 10 passages (>P10). Magnified view is in boxes.

(C) Immunofluorescence (IF) and histological (H&E) analysis of TO phenotype and morphology. Regarding E-cadherin IF (green), arrows indicate outer layer cuboidal epithelium (CE) or inner layer stratified epithelium (SE). Nuclei are counterstained with Hoechst33342 (blue; for all IF images). Delineation between layers is indicated by white dotted line. Black dotted line delineates the centrally localized core region in H&E.

(D) Ultrastructural (TEM) and IF (of indicated markers) characterization of TO. Arrows indicate keratin filaments (left TEM images) or outer basement membrane (right TEM images). Boxed areas are magnified.

(E) Heatmap of gene expression of DE TFs, proposed DESC markers, and EMPs in primary molar and incisor tissue (PD7) and early- and late-passage TOs, as quantified by qRT-PCR analysis. Data are presented as relative expression to Gapdh (ΔCt) and Z score normalized. Colors range from blue (low expression) to yellow (high expression).

(F) IF analysis of SOX2 and AMELX in TOs (green). Boxed areas are magnified.

(G) Bar graph (mean ± standard error of the mean [SEM]) showing expression of Isl1 relative to Gapdh (ΔCt) for primary molar and incisor tissue (left), early-passage TOs (middle), and late-passage TOs (right). Data points represent biological replicates from independently obtained primary tissue and independently established organoid lines; unpaired t test.

(H) IF analysis of ISL1 in TOs and primary molar and incisor. Boxed areas are magnified.

Scale bars: 250 μm for bright-field images, 25 μm for IF and H&E images, and 1 μm for TEM images, unless indicated otherwise. ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S1.