Figure 7.
Effect of AREG on HSC maintenance in other DNA repair–deficient models and aged mice. (A) IR induces persistent DNA damage and high levels of Areg expression in CD45–LepR+ cells from Fancd2–/–, Atm–/–, and aged mice. BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, and aged mice were subjected to 300 cGy IR, followed by flow cytometry–based γ-H2AX analysis (left) (n = 8-9) and qPCR analysis of Areg transcripts (right) (n= 4) at different time points after IR treatment. One-way ANOVA followed by t tests were performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–) separately by comparing the indicated time points (0, 2, 8, 16, and 32-hours after IR) for γ-H2AX MFI and relative Areg for different genotypes (WT, Fancd2–/–, or Atm–/–). Two-tailed unpaired t test or Wilcoxon rank sum test was performed in the indicated groups: young vs old at time points 0, 2, 4, 8, 16, or 32 hours after IR) for relative Areg expression in young and old mice. (B) Progenies from the coculture of WT SLAM cells with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, or aged mice produce a high number of myeloid CFUs. WT SLAM cells were cocultured with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, WT control mice, aged mice, or young control mice for 5 days. One hundred cocultured progenies were subjected to CFU assay (n = 6). One-way ANOVA was performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–) in the left panel. Two-tailed, unpaired t test or Wilcoxon rank sum test was performed in the indicated groups (young vs aged) in the right panel. (C) Progenies from the coculture of WT SLAM cells with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, or aged mice show compromised hematopoietic repopulation. One thousand cocultured progeny cells (CD45.2+) from the coculture described in panel B, along with 2 × 105 radio-protector cells, were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera (CD45.2+) were determined using flow cytometry at different time points after BMT (n = 6). One-way ANOVA, followed by t tests, was performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–), separately comparing them at the indicated time points (4, 8, 12, or 16 weeks after BMT) in the left panel. In the right panel, 2-tailed, unpaired t test or Wilcoxon rank sum test was performed to compare young vs aged at different time points (4, 8, 12, or 16 weeks after BMT). (D) Progenies from coculture with BM CD45–LepR+ from Fancd2–/–, Atm–/–, and aged mice were defective in long-term repopulating. WBMCs from the recipients described in panel C were transplanted into sublethally irradiated BoyJ recipients. Donor-derived chimera (CD45.2+) were determined by flow cytometry 16 weeks after BMT (n = 6-7). One-way ANOVA was performed to compare genotypes (WT, Fancd2–/–, or Atm–/–) in the panel. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare young vs aged in the right panel. (E) Systemic anti-AREG treatment improves HSC function in Fancd2–/–, Atm–/– (left), and aged (right) mice. Mice of the indicated genotypes were treated with 3 doses of anti-AREG or IgG (50 μg each; every other day for 5 days), followed by the analysis of BM cell counts, total HSCs, and mbHSCs at 4 weeks after treatment (n = 6). Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG for each genotype (WT, Fancd2–/–, or Atm–/–) (top) or within each group (young or old) (bottom). (F) Systemic anti-AREG treatment improves long-term repopulating of HSCs from Fancd2–/–, Atm–/– (left), and aged mice (right). One hundred SLAM cells from the mice described in panel E (CD45.2+) was transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera were measured 16 weeks after BMT. Two-tailed, unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG within each genotype (WT, Fancd2–/–, or Atm–/–) (left), or within each group (young or old (right). (G) Genetic knockdown of Areg in Fancd2–/– and Atm–/– MSCs improves the repopulating capacity of progenies from cocultured SLAM cells. MSCs from WT, Fancd2–/–, and Atm–/– mice were transduced with lentiviral particle expressing scramble short hairpin RNA or short hairpin RNA targeting Areg. Progenies of WT LSK cells (CD45.2+) cocultured with sorted GFP+ MSCs, along with 2 × 105 radio-protector cells, were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera (CD45.2+) were measured by flow cytometry at different time points after BMT (n = 6). Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG within each genotype (WT, Fancd2–/–, or Atm–/–). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. shAreg, short hairpin Areg.

Effect of AREG on HSC maintenance in other DNA repair–deficient models and aged mice. (A) IR induces persistent DNA damage and high levels of Areg expression in CD45–LepR+ cells from Fancd2–/–, Atm–/–, and aged mice. BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, and aged mice were subjected to 300 cGy IR, followed by flow cytometry–based γ-H2AX analysis (left) (n = 8-9) and qPCR analysis of Areg transcripts (right) (n= 4) at different time points after IR treatment. One-way ANOVA followed by t tests were performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–) separately by comparing the indicated time points (0, 2, 8, 16, and 32-hours after IR) for γ-H2AX MFI and relative Areg for different genotypes (WT, Fancd2–/–, or Atm–/–). Two-tailed unpaired t test or Wilcoxon rank sum test was performed in the indicated groups: young vs old at time points 0, 2, 4, 8, 16, or 32 hours after IR) for relative Areg expression in young and old mice. (B) Progenies from the coculture of WT SLAM cells with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, or aged mice produce a high number of myeloid CFUs. WT SLAM cells were cocultured with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, WT control mice, aged mice, or young control mice for 5 days. One hundred cocultured progenies were subjected to CFU assay (n = 6). One-way ANOVA was performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–) in the left panel. Two-tailed, unpaired t test or Wilcoxon rank sum test was performed in the indicated groups (young vs aged) in the right panel. (C) Progenies from the coculture of WT SLAM cells with BM CD45–LepR+ cells from Fancd2–/–, Atm–/–, or aged mice show compromised hematopoietic repopulation. One thousand cocultured progeny cells (CD45.2+) from the coculture described in panel B, along with 2 × 105 radio-protector cells, were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera (CD45.2+) were determined using flow cytometry at different time points after BMT (n = 6). One-way ANOVA, followed by t tests, was performed to compare different genotypes (WT, Fancd2–/–, or Atm–/–), separately comparing them at the indicated time points (4, 8, 12, or 16 weeks after BMT) in the left panel. In the right panel, 2-tailed, unpaired t test or Wilcoxon rank sum test was performed to compare young vs aged at different time points (4, 8, 12, or 16 weeks after BMT). (D) Progenies from coculture with BM CD45–LepR+ from Fancd2–/–, Atm–/–, and aged mice were defective in long-term repopulating. WBMCs from the recipients described in panel C were transplanted into sublethally irradiated BoyJ recipients. Donor-derived chimera (CD45.2+) were determined by flow cytometry 16 weeks after BMT (n = 6-7). One-way ANOVA was performed to compare genotypes (WT, Fancd2–/–, or Atm–/–) in the panel. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare young vs aged in the right panel. (E) Systemic anti-AREG treatment improves HSC function in Fancd2–/–, Atm–/– (left), and aged (right) mice. Mice of the indicated genotypes were treated with 3 doses of anti-AREG or IgG (50 μg each; every other day for 5 days), followed by the analysis of BM cell counts, total HSCs, and mbHSCs at 4 weeks after treatment (n = 6). Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG for each genotype (WT, Fancd2–/–, or Atm–/–) (top) or within each group (young or old) (bottom). (F) Systemic anti-AREG treatment improves long-term repopulating of HSCs from Fancd2–/–, Atm–/– (left), and aged mice (right). One hundred SLAM cells from the mice described in panel E (CD45.2+) was transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera were measured 16 weeks after BMT. Two-tailed, unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG within each genotype (WT, Fancd2–/–, or Atm–/–) (left), or within each group (young or old (right). (G) Genetic knockdown of Areg in Fancd2–/– and Atm–/– MSCs improves the repopulating capacity of progenies from cocultured SLAM cells. MSCs from WT, Fancd2–/–, and Atm–/– mice were transduced with lentiviral particle expressing scramble short hairpin RNA or short hairpin RNA targeting Areg. Progenies of WT LSK cells (CD45.2+) cocultured with sorted GFP+ MSCs, along with 2 × 105 radio-protector cells, were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Donor-derived chimera (CD45.2+) were measured by flow cytometry at different time points after BMT (n = 6). Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare IgG vs α-AREG within each genotype (WT, Fancd2–/–, or Atm–/–). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. shAreg, short hairpin Areg.

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