NOTCH2 Promotes Osteoclast Maturation and Metabolism and Modulates the Transcriptome Profile During Osteoclastogenesis

Bone marrow derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice were cultured with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). Bulk RNA-Seq revealed enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis, in Notch2tm1.1Ecan cells. Single cell RNA-Seq data of BMMs treated with M-CSF or M-CSF and RANKL for 3 days identified 11 well-defined cellular clusters. There was an increased number of cells expressing gene markers associated with the osteoclast and with related clusters in Notch2tm1.1Ecan than in control BMMs. To obtain BMMs, the bone marrow from 7 to 8-week-old experimental and control sex-matched littermate mice was removed by flushing. Cells were cultured in a-minimum essential medium (a-MEM) in the presence of 10% fetal bovine serum and M-CSF at 30 ng/ml on uncoated plastic petri dishes, grown, collected and seeded on tissue culture plates in the presence of M-CSF and RANKL. For bulk RNA-Seq, cultures were carried out until multinucleated tartrate resistant acid phosphotase (TRAP)-positive cells were formed. For scRNA-Seq, BMMS were seeded on culture plates coated with Cellmatrix Type I-A Collagen, grown in a-MEM in the presence of either M-CSF at 30 ng/ml alone or M-CSF and RANK-L at 10 ng/ml for 3 days. The collagen gel was digested and cells collected and processed for scRNA-Seq.