Inhibition of AREG by anti–AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2^fl/fl mice rescues HSC defects caused by AREG. (A) Schematic presentation of the experimental design. Systemic treatment of LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice with anti-AREG or IgG every other day for 5 days. Total BM cells and frequencies of total HSCs and mbHSCs were determined 4 weeks after treatment. (B) Systemic treatment of anti-AREG improves BM parameters of LepR-Cre;Brca2^fl/fl mice. LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice were treated with 50 μg anti-AREG or IgG. The numbers of WBMCs/femur (left), HSCs (middle), and mbHSCs (right) cells were determined at 4 weeks after treatment (n = 7-8). Frequencies (left) and absolute numbers (right) are shown. (C) Anti-AREG improves BM niche function in LepR-Cre;Brca2^fl/fl mice. A total of 1 × 10⁴ WT CD45.1⁺ BM LSK cells along with 2 × 10⁵ protector cells (CD45.2⁺) were transplanted into lethally irradiated LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^f/f recipient mice (CD45.2⁺), followed by 3 doses (every other day for 5 days) of 50 μg anti-AREG or IgG treatment 10 days after BMT (top). Donor (CD45.1⁺) cell engraftment (left) (frequencies and absolute numbers) and lineage differentiation (right) were determined by flow cytometry 16 weeks after BMT (bottom) (n = 6). (D) Schematic presentation of experimental design. WT SLAM was cocultured with BM CD45^–LepR⁺ cells isolated from Areg^+/+LepR-Cre;Brca2^fl/fl or Areg^–/–LepR-Cre;Brca2^fl/fl mice, followed by CFU or BMT assay. (E) Deletion of Areg limits expansion of myeloid progenitors. Progenies of WT SLAM cells cocultured with BM CD45^–LepR⁺ cells from Areg^+/+LepR-Cre;Brca2^fl/fl or Areg^–/–LepR-Cre;Brca2^fl/fl mice were subjected to CFU assay (n = 7). (F) Deletion of Areg improves the repopulating capacity of progenies from cocultured SLAM cells. Progenies (CD45.2⁺) described in panel E, along with 2 × 10⁵ radio-protector cells, were transplanted into lethally irradiated BoyJ recipients (CD45.1⁺). Donor-derived chimera (CD45.2⁺) were measured using flow cytometry at different time points after BMT (n = 6). (G) Deletion of Areg improves long-term hematopoietic reconstitution of progenies from cocultured SLAM cells. WBMCs from the primary recipients (CD45.2⁺) described in panel F were pooled and transplanted into sublethally irradiated BoyJ recipients (CD45.1⁺). Donor-derived chimera (CD45.2⁺) were measured using flow cytometry 16 weeks after BMT (n = 6). Frequencies (left) and absolute numbers (right) are shown. Results are presented as mean ± standard deviation of 3 independent experiments. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG within Brca2^+/+ or Brca2^fl/fl groups in panel B and C (left); IgG vs α-AREG within Brca2^+/+ or Brca2^fl/fl groups for different subpopulations (Mac1⁺Gr1⁺, CD3ε⁺ or B220⁺ cells) in panel C (right); Areg^+/+ vs Areg^–/– in panel E and G; Areg^+/+ vs Areg^–/– at different time points (4, 8, 12, and 16 weeks after BMT) in panel F. ∗P < .05; ∗∗P < .01.