Single-cell analysis of the primary BM microenvironment in sick 9p21s^+/− mice. (A) t-Distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) showing 4 niche clusters 11 to 14 in sick 9p21^+/− mice (n = 2) and WT mice (n = 2). (B) Expression level of population marker genes. Acan (cluster 11 = chondrocyte); Cxcl12 (cluster 12 = MSC); Spp1 (cluster 13 = osteolineage cell, osteolineage); and Dcn (cluster 14 = fibroblast). (C) The frequency of clusters in total niche cells. (D) Gene expression changes in the MSC population. Symbols represent single cells (WT cells, n = 134; 9p21^+/− cells, n = 41). (E) Gene expression changes in the chondrocyte population. Symbols represent single cells (WT-Y cells, n = 67; 9p21^+/−-Y cells, n = 105). (F) Gene expression changes in osteolineage cell population. Symbols represent single cells (WT cells, n = 66; 9p21^+/− cells, n = 71). (G) Gene expression changes in fibroblast population. Symbols represent single cells (WT cells, n = 43; 9p21^+/− cells, n = 88). (H) Volcano plot showing the upregulated and downregulated gene expression in sick 9p21^+/− mice total niche cells (pooled clusters 11-14) compared with WT mice total niche cells. The significantly differentially expressed genes were labeled. Data represent the mean ± SD. Statistical significance was defined using Mann-Whitney test and is shown as follows: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 in panels D-G. 9p21^+/− -O, 16-month-old sick mouse; 9p21^+/− -Y, 10-month-old sick mouse; WT-O, 16-month-old WT mouse; WT-Y, 10-month-old WT mouse.