Fig. 4

Reconstruction of bilineage trajectories in NP cells. a UMAP visualization of NPPC, chondrocyte, fibroblast, and osteogenic subclusters. The principal graph of trajectories reported by Monocle 3 was rooted in NPPC-3, as indicated by a cycle. UMAP, Uniform manifold approximation and projection. b Developmental pseudotime for cells present along the trajectory inferred by Monocle 3, with osteogenic and chondrogenic branches coming from NPPC subclusters. c Heatmap showing the scaled mean expression of modules of coregulated genes grouped by Louvain community analysis across the subclusters. d UMAP plots showing the relative expression level of representative gene modules in NP cell subclusters. e Pseudotime kinetics of the indicated genes in the modules in d from the root along the trajectories to chondrogenic and osteogenic differentiation. f Histogram showing the pathways enriched by ReactomePA for each module indicated in d. g Representative alizarin red (top left), oil red O (bottom left), alcian blue (top right), and safranine O/fast green (bottom right) staining after the osteogenic, adipogenic, and chondrogenic differentiation of PROCR⁺ cells (n = 3). Magnified images of the boxed areas are shown on the right. White scale bars, 400 μm. Black scale bars, 200 μm