Fig 1: Distinct subclusters of the osteoblastic lineage in fibroblast cells cluster. A. Left panels: Uniform Manifold Approximation and Projection (UMAP) of 2194 fibroblasts (as in Figure 1A), annotated and colored by the sample type of origin (HCs, PDs, and PDTs) and clustering. Right panels: UMAPs color-coded for expression (gray to red) of key cell-type markers to define the clusters. Red contours (from left to right): Fibroblasts (cluster 1/2/3/5/8/9), Pericytes (cluster 4/7), Myofibroblasts (cluster 6), and Proliferative cell (cluster 10). B. Left panel: UMAP of 1358 fibroblasts (cluster 1/2/3/5/8/9 in A), annotated and colored by clustering. Center panels: violin plots showing distinct expressions of the selected marker genes (Row) in each subcluster. Right panel: identified subpopulations of Fibroblasts (subcluster 1-7) with the percentages shown. PLF: Periodontal Ligament Fibroblasts; MSC: Mesenchymal Stem Cell; pre-OB: pre-Osteoblast; OB: Osteoblast; FB: Fibroblast. C. The box plots showing the percentage of cells for each of seven subclusters as in (B) from HC (blue, n=655), PD (red, n=1095), and PDT samples (green, n=158) with plot center, box, whiskers, and points corresponding to the median, IQR, 1.5 × IQR and >1.5× IQR, respectively. Statistical analysis was performed using unpaired two-tailed t-tests. D. Representative images of in vitro assays to determine multiple differentiation potential of CD55+NT5E+LepR+ cells. Periodontal cells of a clinically healthy donor were collected and stained with various antibodies and subjected to flow cytometry sorting. CD55+NT5E+LepR+ cells were collected and subjected to the following assays. For osteogenic induction, colony-forming units (CFU-F, left)/alkaline phosphatase (ALP)-positive colony-forming units (CFU-ALP, middle)/nodules (right) formation were examined. For adipogenic induction, Oil red staining was performed to examine adipocytes. For chondrogenic induction, Alcian blue staining was performed to examine chondrocytes. E. Representative images of immunofluorescence staining (left panels) and the quantification (right panel) of periodontal tissues from HC (left) and PD samples (right) for double fluorescent analysis of CD55 (green) and LepR (red) expression. Top panel: Merged; bottom left: CD55; bottom right: LepR. Scale bar = 100µm. F. Upper panel: RNA velocity analysis of MSCs (subcluster 2), pre-OBs (subcluster 3), and OBs (subcluster 4) with velocity field projected onto the UMAP plot of fibroblast subclusters from Figure 2B. Arrows show the local average velocity evaluated on a regular grid and indicate the extrapolated future states of cells. Bottom panel: Monocle pseudotime analysis revealing the progression of MSCs (subcluster 2), pre-OBs (subcluster 3), and OBs (subcluster 4). Trajectory reconstruction of all single cells revealing three branches: Pre-branch, Fate 1, and Fate 2. G. Heatmap showing the scaled expression of differently expressed genes in three branches as in (F), cataloged into three major gene clusters (labels on left) that vary as a function of pseudotime, highlighting specific representative genes in each gene cluster along the right margin. From the center to the left of the heatmap, the kinetic curve from the pre-branch along the trajectory to fate 2 branch. From the center to the right, the curve from pre-branch to successful branch. H. GO analysis of differently expressed genes associated with three gene clusters as in (G) identified unique response pathways for each branch.