Interspecies Single‐Cell RNA‐Seq Analysis Reveals the Novel Trajectory of Osteoclast Differentiation and Therapeutic Targets

Abstract

Bone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone-resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor-kappa-Β ligand (RANKL) stimulation. We performed single-cell RNA-sequencing analyses of human and murine osteoclast-lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases.

Advancing single cell technology: iSCseq drives living subcellular transcriptomic profiling in osteoimmune diversity

“…0.3.4 ( 19 ) were used in the integrative analysis of conventional scRNA-seq and iscRNA-seq. Four datasets of scRNA-seq,GSM4419695 (post RANKL stimulation day 0), GSM4419699 (day 1), GSM4419703 (day 3) by Dr. Tsukasaki ( 20 ), and published undeposited data (day 4) by Dr. Omata ( 21 ) were included. Before merging datasets, entrez IDs were transformed to gene symbols with EnsDb.Mmusculus.v79 ( 22 ).…”

“…Conventional scRNA-seq has already been applied for in vitro Osteoclast culture ( 4 ). Our group had described the difference in the OC differentiation pathway between human and mouse ( 5 ). However, conventional Sc techniques does not allow the separation of OCs into Scs due to their large size and fragility.…”

“…To validate our iSCseq approach, we then performed integrative analysis with two previously published scRNA-seq studies of in vitro OC differentiation which used a traditional microfluid-based technique ( 4, 5 ). Five datasets were integrated on a UMAP Fig.…”

“…To clarify the hidden mechanism inside OCs, scRNA-seq was performed for in vitro -cultured OCs 15,16 . However, conventional single-cell techniques theoretically do not allow for the separation of OCs into single-cell suspensions because of their large size and fragility.…”

Single‐Cell RNA‐Sequencing Leading to Breakthroughs in Musculoskeletal Research

“…In the first article by Fujii and colleagues, ( 1 ) the authors identified a novel “surgery‐induced” highly inflammatory CD9 + IL1 + macrophage population in a mouse model of anterior cruciate ligament reconstruction surgery, which resolved early and became replaced by another type of macrophages. In the second article by Omata and colleagues, ( 2 ) the authors identified a GTPase family member, RAB38, as a highly expressed molecule in both human and murine osteoclast precursor cells. This protein was associated with dynamic changes in histone modification and transcriptional regulation.…”