Osteoclast biology in the single-cell era

Abstract

Osteoclasts, the only cells that can resorb bone, play a central role in bone homeostasis as well as bone damage under pathological conditions such as osteoporosis, arthritis, periodontitis, and bone metastasis. Recent studies using single-cell technologies have uncovered the regulatory mechanisms underlying osteoclastogenesis at unprecedented resolution and shed light on the possibility that there is heterogeneity in the origin, function, and fate of osteoclast-lineage cells. Here, we discuss the current advances and emerging concepts in osteoclast biology.

Keywords: Bone metabolism; Osteoblast; Osteoclast; Osteoimmunology; Single-cell analysis.

Fig. 1

Schematic of the osteoclast culture system. Murine bulk bone marrow cells are treated with M-CSF for 2 days, and then, these cells are stimulated with RANKL in the presence of M-CSF. Osteoclasts appear in the culture system after 3–5 days of RANKL stimulation. The osteoclast culture contains heterogeneous populations of cells, only a portion of which is able to differentiate into mature osteoclasts

Fig. 2

Molecular mechanisms underlying osteoclast differentiation. RANKL, the master regulator of osteoclastogenesis, is expressed by osteocytes and Bglap^hi osteoblasts. Dcn^hi osteoblasts locally produce OPG to inhibit osteoclast differentiation and activation. RANKL binding to RANK expressed by osteoclast progenitors results in the activation of signaling cascades including MAPK and NF-κB pathways via TRAF6 and TAK1. The RANKL/RANK signal cooperates with signaling from ITAM-containing immunoglobulin-like receptors such as TREM-2, SIRP ββ, Siglec-15, OSCAR, PIR-A, and FcγRIII. These signaling cascades ultimately lead to the auto-amplification of NFATc1, the master transcription factor of osteoclastogenesis

Fig. 3

Single-cell landscape of osteoclastogenesis. a Representative image of osteoclast differentiation culture system after 3 days of RANKL stimulation in bone marrow cells from CtsK-Cre CAG-CAT-EGFP mouse. The multinucleated giant cells labeled with EGFP are osteoclasts. Most of the cells in the culture system failed to differentiate into mature osteoclasts. Green (EGFP), CTSK; red, actin; blue, DAPI. b The osteoclast differentiation trajectory estimated by pseudotime analysis using the scRNA-seq data obtained from the in vitro osteoclast culture system. c Schematic of the stepwise cell fate decision pathways during osteoclastogenesis unveiled by scRNA-seq

Fig. 4

Emerging mysteries in the osteoclast biology. Single-cell studies have provoked new questions in the osteoclast biology field. Osteoclast precursors may comprise different subsets depending on life stages and pathologies; however, functional difference among osteoclasts derived from distinct precursors remains unclear. Given that osteoclast precursors can fuse one another to differentiate into osteoclasts, it will be important to elucidate the regulatory mechanisms of the multinuclear system to understand the functional diversity of osteoclasts. Although osteoclasts are thought to die quickly by apoptosis after resorbing bone, novel hypotheses regarding the fate of osteoclasts have emerged. Further studies are needed to draw a comprehensive picture of osteoclast life cycle and its functional diversity in health and disease