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Letters to the Editor
ablation decreased the percentage of CD31+c-Kithigh hematopoietic clusters in the E11.5 AGM (0.087±0.005% vs. 0.111±0.006%). Consistently, a greater than 35% reduction in the number of CD31+c-Kithigh cells was observed in the E11.5 Wip1-/- AGM (159±10 vs. 249±14
cells/AGM, respectively) (Figure 3N, O). Moreover, the hematopoiesis-related genes P2-Runx1 (a proximal P2 promoter of Runx1) and Gata2 were reduced significantly after the deletion of Wip1 in the E11.5 AGM clusters but increased in the E11.5 YS (Online Supplementary Figure
ABC
DE
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NOP
Figure 3. Pre-hematopoietic stem cell maturation was impeded by Wip1 ablation in the aorta-gonad-mesonephros region on embryonic day 11.5. (A) Flow cytometric analysis showing the percentages of pre-hematopoietic stem cell (HSC) I (CD31+ CD41lowCD45–) and pre-HSC II (CD31+CD45+) in wild-type (WT) and Wip1-/- aorta-gonad-mesonepros on embryonic day (E)11.5. (B-E) The percentages and absolute numbers of pre-HSC I (B and C, respectively) and pre-HSC II (D and E, respectively), respectively. n=6, **P<0.01. (F, G) The repopulating potential of pre-HSC I (CD31+CD45–) and pre-HSC II (CD31+CD45+) from E11.5 WT and Wip1-/- AGM regions after co-culture with OP9-DL1 cells for 6 days. Chimerism was determined in the peripheral blood at 16 weeks after transplantation. The lines represent average chimerism. (H, I) Transplantation assays showed the engraftment capacity of pre-HSC I (CD31+CD41lowCD45–) and pre-HSC II (CD31+CD45+) of E11.5 WT AGM regions co-cultured with OP9-DL1 with or without CCT007093 (CCT, 25 mM) for 4 days. **P<0.01. (J-M) Donor-derived multi- lineage output of myeloid (Gr-1+/Mac1+, GM), T (CD3+) and B (B220+) cells derived from pre-HSC I (J) and pre-HSC II (K) of E11.5 WT and Wip1-/- AGM region and of E11.5 WT AGM with or without the Wip1 inhibitor, CCT (L and M) at 16 weeks after transplantation. Ctr=dimethlysulfoxide control, *P<0.05, **P<0.01. (N) Representative flow cytometric analysis of CD31+c-Kithigh (CD31+c-Kithi) hematopoietic cluster cells in the E11.5 WT and Wip1-/- AGM region. (O) Percentages and absolute numbers of CD31+c-Kithigh cluster cells (n=9, ***P<0.001). (P) Cell cycle status of endothelial cells (EC, CD31+CD41–CD45–), pre-HSC I (I, CD31+CD41lowCD45–) and pre-HSC II (II, CD31+CD45+) from WT and Wip1-/- AGM regions was examined by Ki67 and 7-AAD staining (n=3, *P<0.05). WT: wild- type; KO: knockout; ee: embryo equivalent; GM: Gr-1+/Mac-1+ cells; T: CD3+ cells; B: B220+ cells.
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