单细胞测序解析下颌骨与肢芽发育异质性

doi:10.13701/j.cnki.kqyxyj.2025.10.004

摘要/Abstract

摘要： 目的: 本研究旨在通过比较小鼠胚胎10.5 d(embryonic day 11.5, E10.5)~15.5 d(E15.5)下颌骨与肢芽的细胞组成,探讨不同组织部位间充质干细胞(mesenchymal stem cells, MSCs)的分化轨迹与细胞功能。方法: 整合文献中小鼠E10.5~E15.5阶段下颌骨及肢芽单细胞转录组RNA测序(single cell RNA sequencing, scRNA-seq)数据集,利用Seurat包进行降维聚类和细胞注释,分离下颌骨间充质干细胞(mandible MSCs, m-MSCs)及肢芽间充质干细胞(limb bud MSCs, l-MSCs)亚集,通过monocle3包构建拟时序分化轨迹,结合差异基因分析及GO与KEGG功能富集分析,系统解析m-MSCs与l-MSCs的基因表达动态与功能差异。提取颌骨骨膜中的m-MSCs和长骨骨膜中的l-MSCs进行体外成骨和成软骨诱导分化实验,qPCR技术评估分化潜能。结果: scRNA-seq分析显示,在骨组织发育早期,m-MSCs(E10.5)与l-MSCs(E11.5)转录组高度相似,仅存在36个差异基因。随着发育进程推移,m-MSCs(E14.5)与l-MSCs(E15.5)的差异基因数量逐渐增加至273个,其中m-MSCs在E10.5~E14.5期间持续表达Prrx1、Cxcl12、Pdgfra等干细胞标志基因,而上述标志基因在E11.5~E15.5阶段的 l-MSCs中明显下降。l-MSCs早期高表达Sox9和Col2a1,富集软骨形成相关通路,而m-MSCs则表现为Runx2提前激活。体外实验证实在基础状态下,成体m-MSCs的Runx2表达水平显著高于l-MSCs,而后者具有较高的Sox9基因表达。结论: 本研究显示不同组织部位的间充质干细胞具有不同的分化命运和细胞功能。m-MSCs相较于l-MSCs具有更强的成骨能力,但其成软骨能力相对较低。

关键词: 干细胞, 间充质干细胞, 颌骨, 肢芽, 发育

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

金思宇, 张聪慧, 纪伟. 单细胞测序解析下颌骨与肢芽发育异质性[J]. 口腔医学研究, 2025, 41(10): 849-855.

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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