Single-Cell Transcriptomic Analysis Reveals Developmental Relationships and Specific Markers of Mouse Periodontium Cellular Subsets

Abstract

The periodontium is essential for supporting the functionality of the tooth, composed of diversity of mineralized and non-mineralized tissues such as the cementum, the periodontal ligament (PDL) and the alveolar bone. The periodontium is developmentally derived from the dental follicle (DF), a fibrous tissue surrounding the developing tooth bud. We previously showed through in vivo lineage-tracing experiments that DF contains mesenchymal progenitor cells expressing parathyroid hormone-related protein (PTHrP), which give rise to cells forming the periodontal attachment apparatus in a manner regulated by autocrine signaling through the PTH/PTHrP receptor. However, the developmental relationships between PTHrP+ DF cells and diverse cell populations constituting the periodontium remain undefined. Here, we performed single-cell RNA-sequencing (scRNA-seq) analyses of cells in the periodontium by integrating the two datasets, i.e. PTHrP-mCherry+ DF cells at P6 and 2.3kb Col1a1 promoter-driven GFP+ periodontal cells at P25 that include descendants of PTHrP+ DF cells, cementoblasts, osteoblasts and periodontal ligament cells. This integrative scRNA-seq analysis revealed heterogeneity of cells of the periodontium and their cell type-specific markers, as well as their relationships with DF cells. Most importantly, our analysis identified a cementoblast-specific metagene that discriminate cementoblasts from alveolar bone osteoblasts, including Pthlh (encoding PTHrP) and Tubb3. RNA velocity analysis indicated that cementoblasts were directly derived from PTHrP+ DF cells in the early developmental stage and did not interconvert with other cell types. Further, CellPhoneDB cell-cell communication analysis indicated that PTHrP derived from cementoblasts acts on diversity of cells in the periodontium in an autocrine and paracrine manner. Collectively, our findings provide insights into the lineage hierarchy and intercellular interactions of cells in the periodontium at a single-cell level, aiding to understand cellular and molecular basis of periodontal tissue formation.

Plap-1/Aspn lineage tracing and single-cell transcriptomics reveals cellular dynamics in the periodontal ligament

“…5J and S5F). However, the cluster did not specifically express recently reported markers (Nagata et al 2021), including Parathyroid hormone-related protein ( Pthlh), Class III β-tubulin ( Tubb3) , and Wnt inhibitory factor 1 (Wif1) (Fig. S5G).…”

“…However, the exact cellular dynamics of the cementoblasts were to be investigated with definitive markers. Recent analyses of cementum development in mice have highlighted several molecules that are specifically expressed in cementoblasts (Nagata et al 2021). In this study, we defined cementoblasts as Plap-1 - Ibsp + Sparcl1 + and alveolar osteoblasts as Plap-1 - Ibsp + Col11a1 + .…”

Periodontal tissue stem cells and mesenchymal stem cells in the periodontal ligament

“…More than half of the cells analyzed were epithelial and endothelial cells; the mesenchymal cells were divided into MSCs, fibroblasts, and Schwann cells [74] . Nagata et al isolated cells from molars from 25-day-old Col1a1-GFP mice and performed a scRNA-seq analysis, which yielded an Scleraxis-positive PDL cell population and identified molecules specific to cementoblast or osteoblast; Pthlh , Tubb3 , and Wif1 as cementoblast-specific molecules and Phex , Nfib , and Pthr1r as osteoblast-specific molecules [75] . However, the development of PDL is usually not complete for PDL at 25 days old, and whether these results can be adapted for adult PDL, cementoblasts, and osteoblasts remains to be investigated.…”

Diverse stem cells for periodontal tissue formation and regeneration

“…This approach has been extensively applied to tooth‐related cell types (Chiba et al, 2020; Krivanek et al, 2020; Takahashi et al, 2019). We recently reported the developmental relationships between PTHrP + DF cells and their descendants in the periodontium of mouse molars by integrating two scRNA‐seq datasets, (1) PTHrP‐mCherry + DF cells harvested from PTHrP‐mCherry knock‐in mice molars at P6 (GSE120108), and (2) Col1a1‐GFP + mesenchymal periodontal cells harvested from Col1a1(2.3 kb)‐GFP; PTHrP‐creER; R26R‐tdTomato molars at P25 (GSE168450) (Nagata, Chu, Ono, Welch, & Ono, 2021). Our integrative scRNA‐seq analysis identified a cementoblast‐specific metagenes that discriminate cementoblasts from alveolar bone osteoblasts including PTHrP.…”

“…Further characterization of mesenchymal progenitors in the PDL is required to define the function of these stem cells in both mice and humans. Together with mouse genetic approaches, scRNA‐seq analysis has been extensively applied in recent years to reveal the cellular heterogeneity of dental tissues including the periodontium in both mice and humans at a single‐cell level (Chiba et al, 2020; Chiba et al, 2021; Jones et al, 2019; Krivanek et al, 2020; Nagata et al, 2021; Pagella, de Vargas Roditi, Stadlinger, Moor, & Mitsiadis, 2021; Takada et al, 2022; Takahashi et al, 2019; J. Zhao et al, 2021). Moreover, spatial transcriptomic approaches are currently developed to compensate for the disadvantage of conventional RNA‐sequencing approaches that do not allow to retain positional information of each individual cells (Rodriques et al, 2019; Ståhl et al, 2016; Vickovic et al, 2022).…”