Fig 1: VD3 induces Trem2+ macrophage subpopulations in vitro.(A) Experimental workflow. BM was isolated from C57/BL6 mice, and cells were differentiated into “M1” macrophages for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) according to standard protocols (Materials and Methods). The differentiated cells were then either stimulated with 1,25-dihydroxycholecalciferol (active form of VD, VD3) for 24 hours or left untreated, and both populations were analyzed using scRNA-seq. (B) UMAP embedding of cells colored by group. Co-Mac, control macrophages; VD-Mac, VD3-treated macrophages. (C) Cells from cluster 0 from the in vivo monocyte lineage subset (Fig. 3E) were bioinformatically integrated with data from cultured macrophages. hSTSG-Mac, cluster 0 macrophages (Trem2+) from the in vivo dataset. Trajectories (highlighted by orange dotted lines) were determined using Monocle 3. (D) Root cells identified by RNA velocity analysis. (E) Main gene-averaged flow visualized by velocity streamlines and projected onto the UMAP embedding of the integrated dataset. (F) Cells colored by pseudotime using Monocle 3. Black nodes represent the branch points of the trajectories; white node represents the root node. (G) Violin plots showing Trem2, Mertk, and Ccr2 expression. Cells are colored by experimental group. (H) Expression of Trem2, Mertk, Vegfa, and Ccr2 along pseudotime. Cells are colored by experimental group.