Alterations to the bone marrow microenvironment due to free heme exposure. C57BL/6 mice were injected with 50 µmol/kg hemin 3 times per week or phosphate-buffered saline (control) for 30 days to mimic long-term heme exposure. To examine if TLR4 inhibition can block heme-mediated effects in stromal cells, B6 mice were treated with hemin plus TAK-242 (H + T). An additional group of TLR4 KO mice were treated with hemin for comparison. (A) Peripheral blood hematopoietic stem and progenitor cells marked by Lineage-Ckit⁺Sca1⁺Flt3^– expression as measured by flow cytometry represented as fold change in number per mL blood relative to control (N = 3-4). Gene expression of Cxcl12 (B) and Scf (C) among bone marrow MSCs in each group as measured by RT-PCR and normalized to Gapdh (N = 4-8). (D) Mean fluorescence intensity (MFI) of DCFDA among CD45^–Ter119^–CD31^–PDGFRα⁺CD51⁺ MSCs in the bone marrow (N = 3-4 mice). (E) MFI of DCFDA among Lin^–Sca1⁺c-Kit⁺CD48^–CD150⁺ HSCs in the bone marrow (N = 3-4 mice). Data are presented as mean ± standard error of the mean. Data were analyzed with 1-way analysis of variance with Tukey multiple comparison test (A-C), or a 2-tailed, unpaired Student t test (D-E). *P ≤ .05, **P ≤ .01, ***P ≤ .001.