Single-cell RNA sequencing analysis of the temporomandibular joint condyle in 3 and 4-month-old human embryos

Abstract

Background: The temporomandibular joint (TMJ) is a complex joint consisting of the condyle, the temporal articular surface, and the articular disc. Functions such as mastication, swallowing and articulation are accomplished by the movements of the TMJ. To date, the TMJ has been studied more extensively, but the types of TMJ cells, their differentiation, and their interrelationship during growth and development are still unclear and the study of the TMJ is limited. The aim of this study was to establish a molecular cellular atlas of the human embryonic temporomandibular joint condyle (TMJC) by single-cell RNA sequencing, which will contribute to understanding and solving clinical problems.

Results: Human embryos at 3 and 4 months of age are an important stage of TMJC development. We performed a comprehensive transcriptome analysis of TMJC tissue from human embryos at 3 and 4 months of age using single-cell RNA sequencing. A total of 16,624 cells were captured and the gene expression profiles of 15 cell clusters in human embryonic TMJC were determined, including 14 known cell types and one previously unknown cell type, "transition state cells (TSCs)". Immunofluorescence assays confirmed that TSCs are not the same cell cluster as mesenchymal stem cells (MSCs). Pseudotime trajectory and RNA velocity analysis revealed that MSCs transformed into TSCs, which further differentiated into osteoblasts, hypertrophic chondrocytes and tenocytes. In addition, chondrocytes (CYTL1^high + THBS1^high) from secondary cartilage were detected only in 4-month-old human embryonic TMJC.

Conclusions: Our study provides an atlas of differentiation stages of human embryonic TMJC tissue cells, which will contribute to an in-depth understanding of the pathophysiology of the TMJC tissue repair process and ultimately help to solve clinical problems.

Keywords: Cellular transcriptomics; Gene expression; Human embryonic cells; TMJC.

Fig. 1

Overview of the scRNA sequencing of human embryonic TMJC. a The 15 cell clusters identified in human embryonic TMJC using UMAP. b The cell clusters were identified in 3 and 4-month-old human embryonic TMJC. c The expression of CYTL1 and THBS1 in each cluster of human embryonic TMJC. d The immunofluorescent staining of CYTL1 and THBS1 in 3 and 4-month-old human embryonic TMJC, Scale bar = 1 mm and 50 µm

Fig. 2

Subpopulation analysis of TSCs. a The five subpopulations of TSCs were visualized using UMAP. b The cell proportions of subpopulations in TSCs. c The expression of CAPN6 in each cluster of human embryonic TMJC. d The expression of PTN in each cluster of human embryonic TMJC. e Immunofluorescence staining of CAPN6 and PTN in 3 and 4-month-old human embryonic TMJC, Scale bar = 1 mm and 50 µm

Fig. 3

The differentiation relationship analysis of MSCs, TSCs, tenocytes, hypertrophic chondrocytes and osteoblasts. a The differentiation relationship among MSCs, TSCs, tenocytes, hypertrophic chondrocytes and osteoblasts based on Monocle3 analysis. b The differentiation relationship among these five clusters based on RNA velocity analysis. c The expression of key genes including RUNX2, SOX9, MMP13, SOST, VEGFA, COL10A1, MAF, CCDC80 and SYNE2 among the five clusters

Fig. 4

CellChat analysis of human embryonic TMJC clusters. a The number of ligand-receptor pairs in 15 cell clusters. b FGF signaling pathway networks among 15 cell clusters. c The contribution of each ligand-receptor pairs of FGF signaling pathway. d FGF7-FGFR1 signaling pathway networks among 15 cell clusters. e The analysis of outgoing communication patterns of secreting cells and incoming communication patterns of target cells among 15 cell clusters

Fig. 5

Transcription factors analysis among human embryonic TMJC clusters. a The regulon activity analysis showed the on/off of the specific regulons in each cluster. b Heatmap showed the common regulons were turned on between MSCs and TSCs. c Modules analysis of cell clusters and common regulons. d Interaction mapping of transcription factors networks showed common regulons between MSCs and TSCs

Fig. 6

Overview of the cell types and differentiation relationship among human embryonic TMJC cells