Single-cell RNA sequencing analysis of the temporomandibular joint condyle in 3 and 4-month-old human embryos
Background The temporomandibular joint (TMJ) is a complex joint consisting of the condyle, the temporal articu_x0002_lar 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 temporomandibu_x0002_lar 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(3M,4M) 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 mesen_x0002_chymal 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 (CYTL1high+ THBS1high) 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. Overall design: This study utilized single-cell RNA sequencing (scRNA-seq) to analyze the embryonic temporomandibular joint condyle (TMJC) at different developmental stages, including 3M,4M. TMJC tissues were obtained from human embryos, preserved in a tissue preservation solution (Singleron Biotechnologies, Nanjing, China), and transported under cold chain conditions. The tissues were cut into 2–3 mm sections, rinsed with Hanks' Balanced Salt Solution (HBSS), and enzymatically digested for 15 minutes using a tissue dissociation solution (Singleron Biotechnologies, Nanjing, China). The resulting cell suspension was filtered through a 40µm sterile filter (Corning, NY, USA) and centrifuged at 150 g for 5 minutes. Erythrocytes were removed using a lysis reagent (Singleron Biotechnologies, Nanjing, China) for 10 minutes. The cells were resuspended in phosphate-buffered saline (PBS), stained with trypan blue (T6146, Sigma, Burlington, VT, USA), and assessed for viability using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA). The cell suspension was adjusted to a concentration of 1×105 cells/mL, loaded into a microfluidic chip, and single-cell RNA libraries were constructed using the Single-Cell RNA Library Kit (Singleron Biotechnologies, Nanjing, China). The prepared libraries were then sequenced on an Illumina NovaSeq 6000 platform to obtain high-throughput single-cell transcriptomic data.