A systematic dissection of human primary osteoblasts in vivo at single-cell resolution.

Yun Gong,Yuntong Bai,Yusheng Li,Yu Chen,Jie Xie,Xuecheng Yang,Ying Liu,Yihe Hu,Jonathan Greenbaum,Yiping Chen,Hui Shen,Xiaohua Li,Xiang Qiu,Zun Wang,Liang Cheng,Huixi Zhang,Yuping Wang ,Chaohui Zhou ,Lijun Tan ,Jin Yang ,Hong‐Mei Xiao ,Hong‐Wen Deng

doi:10.18632/aging.203452

Abstract

Human osteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. However, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts from one 31-year-old male with osteoarthritis and osteopenia after hip replacement. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was found to be the major source of the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2) in primary osteoblasts, and the expression of NR4A1 was confirmed by immunofluorescence staining on mouse osteoblasts in vivo. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human and mouse osteoblasts, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level.

Highlights

Osteoblasts, which account for 4-6% of resident cells in bone, are derived from multipotent skeletal stem cells (SSCs) through the activation of signaling pathways regulated by osterix (OSX) and Runt-related transcription factor 2 (Runx-2) [1]
Compared with t-SNE [24], another commonly used dimension reduction method in single cell analysis, uniform manifold approximation and projection (UMAP) provides the useful and intuitively pleasing feature that it preserves more of the global structure [23], which is important to identify the systematic differences of gene expression patterns between different clusters through the inter-cluster distances
For the first time, we classified the freshly isolated primary osteoblasts from human bone into three subpopulations based on systematic differences in gene expression profiles and revealed their distinct functional roles in bone metabolism as well as in the regulation of angiogenesis and hemopoiesis

Summary

Introduction

Osteoblasts, which account for 4-6% of resident cells in bone, are derived from multipotent skeletal stem cells (SSCs) through the activation of signaling pathways regulated by osterix (OSX) and Runt-related transcription factor 2 (Runx-2) [1]. Many osteoblasts differentiate into osteocytes, which become embedded in the bone matrix to form a communication network for the regulation of bone formation and resorption. The osteoblasts and osteocytes regulate the differentiation of osteoclasts, which are primarily involved in bone resorption activities [6]. Osteoblasts can promote osteoclast proliferation by producing macrophagecolony stimulating factor (M-CSF) [7]. Osteoblasts can produce the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) to modulate osteoclast proliferation through the RANKL/RANK/OPG pathway [8]. Osteoclasts may regulate bone formation by osteoblasts [9]. The complex dynamics between the major bone cells control the delicate balance of bone formation/resorption that is critical for maintaining bone health

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Results

Conclusion