Figure 1: TIE2-expressing bone marrow endothelial cells produce EGF, and EGF mediates HSC regeneration after irradiation.

(a) Mean numbers (± s.e.m.) of total cells (left), CFCs (middle) and CFU-S12 (right) at day 7 from noncontact cultures of 300 cGy–irradiated bone marrow KSL cells with FL/– bone marrow endothelial cells compared to culture with FL/+ bone marrow endothelial cells or cytokines alone (TSF). Input, population of KSL cells at day 0. *P = 0.003, ^P = 0.04 compared to TSF and FL/+, respectively, for total cells (n = 3–7 experiments per condition); *P < 0.0001, ^P < 0.0001 compared to TSF and FL/+, respectively, for CFCs (n = 3 experiments per condition, t test); *P = 0.04, ^P = 0.02 compared to TSF and FL/+, respectively, for CFU-S12 (n = 3–5 experiments per condition). (b) Left, concentrations of EGF in the bone marrow serum of Tie2Cre; Bak1^−/−; Bax^flox/– (red line), Tie2Cre; Bak1^−/−; Bax^flox/+ (blue line) and C57BL/6 mice (black line) before irradiation (Nonirrad) and at 6 h and 7 d after 750-cGy irradiation. *P = 0.02, **P = 0.04, ***P = 0.04 compared to Tie2Cre; Bak1^−/−; Bax^flox/+ mice determined by one-tailed t test. n = 3 mice per condition; data are shown as the means ± s.e.m. Right, EGF expression in FL/–, FL/+ and C57BL/6 (BL/6) endothelial cells by quantitative RT-PCR. *P = 0.002, ^P = 0.003 compared to BL/6 and FL/+ endothelial cells, respectively, determined by two-tailed t test. Data are shown as the means ± s.e.m. n = 3 experiments per group. (c) Left, representative EGFR surface expression on bone marrow Lin⁺, Lin⁻ and KSL cells from C57BL/6 mice. Right, representative EGFR surface expression on nonirradiated bone marrow SLAM⁺ KSL cells and at 4 h after 700-cGy irradiation. The numbers shown indicate the pergentage of EGFR surface expression on the indicated cell population. (d) Left, EGFR surface expression in each bone marrow subset. *P = 0.009, ^P < 0.001 for bone marrow KSL cells compared to bone marrow Lin⁺ and Lin⁻ cells, respectively. *P = 0.02, ^P = 0.004 for bone marrow SLAM⁺ KSL cells compared to Lin⁺ and Lin⁻ cells, respectively (n = 3–9 experiments per group). Data are shown as the means ± s.e.m. Right, EGFR expression in nonirradiated bone marrow SLAM⁺ KSL (red line), KSL (blue line), Lin⁻ (black line) and Lin⁺ cells (green line, along x axis) and at 4 h after 300-cGy and 700-cGy TBI. *P = 0.002 for the difference in SLAM⁺ KSL cells between the 300 cGy–irradiated and nonirradiated groups, *P = 0.001 for the difference between the 700 cGy–irradiated and nonirradiated groups (n = 3–7 experiments per group). Data are shown as the means ± s.e.m. (e) Representative FACS analyses of phosphorylation of EGFR at Tyr1173 (pEGFR) in bone marrow KSL (left) and bone marrow SLAM⁺ KSL (middle) cells in serum-free culture (blue curve) or in the same conditions plus EGF (red curve). Right, pEGFR in bone marrow KSL cells in mice treated with EGF (red curve) or saline (blue). Isotype-stained cells are shown in black. Percentages of EGFR phosphorylation are shown for each group at right. *P = 0.008 for EGF compared to control cultures of KSL cells, *P < 0.001 for EGF compared to control cultures of bone marrow SLAM⁺ KSL cells (n = 5 experiments per group). Data are shown as the means ± s.e.m. *P = 0.03 for EGF compared to saline treatment of KSL cells in vivo (n = 4 experiments per group). (f) CFC and CFU-S12 content of cultures of irradiated bone marrow KSL cells with TSF or TSF plus EGF. *P = 0.0002, *P = 0.0003 for CFCs (n = 8 experiments per group) and CFU-S12 (n = 9–12 experiments per group), respectively, compared to TSF alone. Data are shown as the means ± s.e.m. (g) Peripheral blood donor CD45.1⁺ cell, myeloid (Mac-1/Gr-1), B cell (B220) and T cell (Thy1.2) engraftment at 8 weeks after transplant in CD45.2⁺ mice after transplantation of irradiated, TSF-cultured CD34⁻KSL cells or irradiated, TSF- and EGF-cultured CD34⁻KSL cells (n = 7–9 mice per group). *P = 0.002 for CD45.1⁺ engraftment, *P = 0.002 for myeloid engraftment. Right, total peripheral blood CD45.1⁺ cell engraftment over time (red line, TSF plus EGF culture; black line, TSF alone). *P = 0.002. Data are shown as the means ± s.e.m. (h) Numbers of KSL cells and CFU-S12 from cultures of bone marrow KSL cells with TSF or TSF plus EGF. *P = 0.03, *P = 0.004. Data are shown as the means ± s.e.m. n = 4–6 experiments per group. Right, percentage donor CD45.1⁺ cell engraftment at 12 weeks in mice transplanted with nonirradiated and TSF-cultured CD34⁻ KSL cells or nonirradiated and TSF- and EGF-cultured CD34⁻ KSL cells. *P = 0.04 determined by two-tailed t test. Data are shown as the means ± s.e.m. n = 4–5 mice per group. A Mann-Whitney test was used for all statistical analyses unless otherwise noted.