Figure 2

Single-cell analysis reveals differential stages of in vitro human osteoclastogenesis. (A) Schematic overview of experimental approach. Specifically, human CD14⁺CD16⁻ monocytes were isolated from PBMCs and transduced with lentivectors encoding the active NOTCH1-ΔE isoform or empty vector as control. Transduced GFP⁺ cells were in vitro stimulated and expanded with RANKL and M-CSF for 5 days and subsequently purified by FACS-sorting for performing single cell RNA-Sequencing (scRNA-Seq) and Ab-sequencing (AbSeq) assays. (B) UMAP plots based on the scRNA-Seq and AbSeq data showing the distribution of Notch1-transduced cells in red vs. control cell subsets in light blue. Each dot represents one cell. (C) UMAP plot as in Panel B, but with each cluster depicted in a different color as determined by the Leiden graph-clustering method (resolution = 1.4). There was a total of 9 different phenotypic clusters identified by the Leiden algorithm. (D) Cumulative percentage of Notch1-transduced and control cells according to each sample group. (E) UMAP plot as in Panel B, but with cells colored based on their assigned MetaCluster (MC) as determined by the Leiden graph-clustering method (resolution = 0.4). (F) Cumulative percentage of Notch1-transduced and control cells according to each MetaCluster. (G) The differentiation and developmental trajectory of human CD14⁺CD16⁻ monocytes following 5 days of in vitro RANKL-stimulation as determined by partition-based graph abstraction (PAGA). Arrow and black bars indicate the direction and the average velocity of the differentiation and progression of each MetaCluster, respectively. (H,I) Diffusion pseudotime plots depicting each MetaCluster in all cells (H) as well as in the Notch1-transduced and control cell subsets (I).