Single-cell Sequencing Resolves Heterogeneity in Mandible and Limb Bud Development

doi:10.13701/j.cnki.kqyxyj.2025.10.004

Abstract

Abstract: Objective: To investigate the differentiation trajectories and cellular functions of mesenchymal stem cells (MSCs) in different tissue sites by comparing the cellular composition of mandible and limb buds of mouse embryos from embryonic day 10.5 (E10.5) to day 15.5 (E15.5). Methods: Single-cell RNA sequencing (scRNA-seq) datasets of mandible and limb buds from mouse E10.5 to E15.5 were integrated from literature. The Seurat package was used for dimensionality reduction, clustering, and cell annotation to isolate mandible MSCs (m-MSCs) and limb bud MSCs (l-MSCs) subsets. Pseudotime differentiation trajectories were constructed using the monocle3 package. Differential gene analysis, along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses, were employed to systematically analyze the dynamic gene expression and functional differences between m-MSCs and l-MSCs. MSCs from the mandibular periosteum (m-MSCs) and long bone periosteum (l-MSCs) were extracted and subjected to in vitro osteogenic and chondrogenic induction differentiation experiments, with their differentiation potential assessed by qPCR. Results: The scRNA-seq analysis revealed that during early bone tissue development, m-MSCs (E10.5) and l-MSCs (E11.5) exhibited highly similar transcriptomes, with only 36 differentially expressed genes (DEGs). As development progressed, the number of DEGs between m-MSCs (E14.5) and l-MSCs (E15.5) increased to 273. Notably, m-MSCs continuously expressed stem cell marker genes (e.g., Prrx1, Cxcl12, Pdgfra, and Cd200) from E10.5 to E14.5, while these markers significantly declined in l-MSCs from E11.5 to E15.5. In the early stages, l-MSCs exhibited high expression of Sox9 and Col2a1, with enrichment of chondrogenesis-related pathways, whereas m-MSCs demonstrated precocious activation of Runx2. In vitro experiments confirmed that under basal conditions, adult m-MSCs showed significantly increased Runx2 expression compared to l-MSCs, while the latter displayed elevated Sox9 expression. Conclusion: The MSCs from different tissue sites exhibited different cellular fate determination and differentiation capacities. Particularly, the m-MSCs demonstrated relatively stronger osteogenic capacity compared to l-MSCs.

Key words: stem cells, mesenchymal stem cells, mandible, limb bud, development

JIN Siyu, ZHANG Conghui, JI Wei. Single-cell Sequencing Resolves Heterogeneity in Mandible and Limb Bud Development[J]. Journal of Oral Science Research, 2025, 41(10): 849-855.

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