Fibrosis-driving cells are transcriptionally reprogrammed in early stages of BM fibrosis and exhibit induction of inflammatory pathways. HSPCs (ckit⁺) were transduced with ThPO-overexpression cDNA or EV and cocultured for 72 hours with primary Gli1⁺ stromal cells (TdTomato⁺; n = 3 per group). Ckit⁺ HSPCs and Gli1⁺ stromal cells were then sort-purified as GFP⁺ and lin^–GFP^-tdTomato⁺, respectively. To analyze fibrosis in vivo, bigenic Gli1CreERt2;tdTomato mice were lethally irradiated 10 days after the last tamoxifen dose and received c-kit–enriched HSPCs from WT littermates expressing either ThPO cDNA (n = 5, 3 male mice) or control cDNA (control, n = 5, 3 male mice). Mice were euthanized at 70 days after transplantation. Gli1⁺ cells were sort-purified as lin^–GFP⁺ tdTomato⁺ and subjected to RNA sequencing. (A) Principal component analysis and heatmap representation with hierarchical clustering of HSPCs transduced with either ThPO or EV control. (B) Principal component analysis and heatmap representation with hierarchical clustering of sort-purified Gli1⁺ stromal cells previously cocultured with either ThPO- or EV-transduced HSPCs. (C) PROGENy pathway analysis of sort-purified HSPCs transduced with ThPO-overexpressing vector (BM fibrosis induction) in a coculture setting with Gli1⁺ stromal cells (coculture HSPCs, left panel), sort-purified Gli1⁺ stromal cells with previous short exposure to ThPO (or EV) HSPCs (Gli1⁺ cells “early,” middle panel), and sort-purified Gli1⁺ stromal cells isolated from a ThPO-overexpression murine model with severe BM fibrosis (fibrosis in vivo, Gli1⁺ cells, right panel). All samples are compared with their respective EV control. (D) NABA-matrisome associated genes in HSPCs (left panel), primary Gli1⁺ stromal cell from coculture (middle panel), and primary Gli1⁺ stromal cells from murine model of ThPO-induced BM fibrosis (right panel). Curved lines on the volcano plots are asymptotic to a value of P = .05 (-log) and a fold-change = .10 of the maximum observed fold change in the signature.