Yoshioka Hirotaka, Okita Saki, Nakano Masashi, Minamizaki Tomoko, Nubukiyo Asako, Sotomaru Yusuke, Bonnelye Edith, Kozai Katsuyuki, Tanimoto Kotaro, Aubin Jane E, Yoshiko Yuji
Department of Calcified Tissue Biology, Graduate School of Biomedical and Health Sciences Hiroshima University Hiroshima Japan.
Department of Anatomy School of Medicine, International University of Health and Welfare Chiba Japan.
JBMR Plus. 2021 May 17;5(6):e10496. doi: 10.1002/jbm4.10496. eCollection 2021 Jun.
The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus-positive (Venus) cells expressing the fluorescent protein Venus under the control of the 2.3-kb promoter were isolated from newborn mouse calvariae and subjected to single-cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus single cells indicated that Venus cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster-specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co-expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed and with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone-forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
目前关于成骨细胞命运的范式是，大多数成骨细胞会经历凋亡，而一些会进一步分化为骨细胞，其他的则会变扁平并作为骨衬细胞覆盖骨表面。已有研究表明，成骨细胞在转录和蛋白质水平上表现出多种成骨细胞标志物的异质性表达。为了进一步探究这种异质性及其生物学意义，从新生小鼠颅骨中分离出在2.3 kb启动子控制下表达荧光蛋白金星（Venus）的金星阳性（Venus）细胞，并进行单细胞RNA测序。对272个Venus单细胞中表达的基因进行功能注释表明，Venus细胞是成骨细胞，可分为四个簇。其中，三个簇（簇1至3）在成骨细胞标志物的表达上表现出相似性，而一个簇（簇4）则明显不同。我们总共鉴定出1920个簇特异性基因，基于既定概念和已知标志物的伪时间排序分析表明，簇1至3捕获了不同成熟阶段的成骨细胞。基因共表达网络分析表明，参与蛋白质合成以及内质网（ER）和高尔基体之间蛋白质运输的基因在这些簇中活跃。然而，这些簇中的细胞也由基因表达的广泛异质性所定义，与成熟阶段无关。簇4的细胞表达成骨细胞标志物的水平相对较低，这表明这种细胞类型不同于活跃形成骨的成骨细胞，保留或重新获得了祖细胞特性。基于对单个成骨细胞转录组的表达和机器学习分析，我们还鉴定出了可能用作成骨细胞成熟阶段新标志物的基因。综上所述，我们的数据表明成骨细胞的异质性比以前记录的要广泛得多，基因谱支持成骨细胞功能活动和发育命运的多样性。© 2021作者。由Wiley Periodicals LLC代表美国骨与矿物质研究学会出版。
