Fig. 2: Gingival keratinocyte diversity is molecularly defined, spatially distinct, and preserved in vitro.

a To validate keratinocyte heterogeneity, healthy human gingival tissues were preserved on the tooth surface after extraction and fixed. b IHC revealed the gradual transition from tooth-facing JKs and SKs to GM, AG, and AM keratinocytes using KRT19; c KRT19-high and -low epithelial stem cells proliferate in the basal layer in health. d All keratinocytes (KRT14⁺) were subclustered from the integrated periodontitis meta-atlas (30 samples, 8584 cells) and assigned annotations (e) based on Louvain clustering. Cell signatures for these populations are plotted and included in Supplementary Data 1. f Using these signatures, we used a custom 12-plex ISH panel to reveal heterogeneity in keratinocyte populations (AG, GM, and JK here; SK and AM as in Fig. 1). Markers such as CXCL14 and NEAT1 marked the AG basal epithelium in the opposite pattern of KRT19 protein of the SK/JK cells; ODAM, RHCG, IL18, and SAA1/2 marked SK and JK cells in sequencing and in situ (See Supplementary Fig. 3 for sulcular, marginal, and alveolar mucosa imaging). g Primary human gingival keratinocytes were cultured over multiple passages. KRT19-high (marked by+) basal and larger suprabasal keratinocytes are found in mixed populations at (h) first passage and over (i) multiple passages. j Using RNA ISH and additional markers, cell subpopulations that were defined in vivo such as AG (LGR6⁺) and SK/JK (FDCSP⁺) can be identified in vitro, suggesting a heterogeneous 2D model of tooth-facing and oral-facing keratinocytes can be utilized for future assays and that these markers are more likely cell identities than cell states. Abbreviations: P Passage; see Fig. 1 legend. Sequential sections from samples were used (n = 3 health). Scale bars: b 100 μm; c 50 μm; f 25 μm; i, j 10 μm. Illustration from (g) created with BioRender.com. For this figure, n = 30-sample, 8554-cell for scRNAseq; n = 3 for tissues; n = 2 for unique primary cell lines.