Abstract
ABSTRACTThe 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 Col1a1 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 Cd34 and Cxcl12 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. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
Highlights
Bone marrow stromal cells (BMSCs; aka mesenchymal stem cells) have capacity to differentiate into multiple cell types including osteoblasts, adipocytes, and chondrocytes.[1,2] Osteoblast lineage fate decision is driven by the master transcription factor RUNX2,(3) which directly regulates the expression of SP7, a transcriptional activator for osteoblast differentiation, resulting in recruitment of SP7 and co-factor DLX to osteoblast enhancers to promote the expression of osteoblast-specific genes.[4,5] Osteoblasts play a pivotal role in bone formation by producing and secreting bone matrix components and initiating matrix mineralization
In calvaria cell cultures fractionated by sequential enzymatic digestions, Venus+ cells were enriched in fractions 5 to 8 (Fig. S1A)
Dimension reduction methods (UMAP and t-distributed stochastic neighbor embedding (t-SNE) analyses) indicated that the 272 Venus+ osteoblasts could be classified into four clusters
Summary
Bone marrow stromal cells (BMSCs; aka mesenchymal stem cells) have capacity to differentiate into multiple cell types including osteoblasts, adipocytes, and chondrocytes.[1,2] Osteoblast lineage fate decision is driven by the master transcription factor RUNX2,(3) which directly regulates the expression of SP7, a transcriptional activator for osteoblast differentiation, resulting in recruitment of SP7 and co-factor DLX to osteoblast enhancers to promote the expression of osteoblast-specific genes.[4,5] Osteoblasts play a pivotal role in bone formation by producing and secreting bone matrix components and initiating matrix mineralization. Single cell colony assays[12] and in situ hybridization and immunohistochemical analyses[13] of osteoblast marker genes have suggested that osteoblasts comprise molecularly heterogeneous populations, which may reflect molecular diversity and functional diversity in osteoblasts.[7,12,13] Among the many facets of cellular heterogeneity, non-genetic (phenotypic) heterogeneity is increasingly being appreciated as not just noise or technical artifact but as a fundamental intrinsic condition for the evolution of organismal robustness, and for the relationship between genetic and developmental robustness, including multipotency and cell type diversification.[14,15] Until recently, analytic tools for transcriptomics were reliably applied mainly to bulk cell samples, but newer technological breakthroughs allow for transcriptomic analysis at the single-cell level.[16,17] In this study, we sought to demonstrate heterogeneity in osteoblasts isolated from calvariae of newborn mice expressing the fluorescent protein Venus under the control of the 2.3-kb Col1a1 promoter (Venus+ osteoblasts) by singlecell transcriptome analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
