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Letters to the Editor
(CCT007093, CCT) was added. None of the recipients that received pre-HSC I cultures with inhibitor was repopulated, whereas six of seven recipients given dimethylsulfoxide (DMSO) instead of the inhibitor achieved chimerism (33.3±9.0%). In the pre-HSC II cul- tures, the presence of the CCT inhibitor reduced the abil- ity of engraftment of pre-HSC II-derived HSC cultures compared to the cultures with DMSO (5/10 [50%] vs. 7/9 [77.8%]; chimerism 13.5±5.2% vs. 46.2±8.8%, respec-
tively) (Figure 3H, I). Additionally, the lymphoid lineage output of pre-HSC II cultures was changed in the pres- ence of the Wip1 inhibitor (Figure 3L, M). These results, together with the direct transplantation findings, indicate that Wip1 educates the maturation of pre-HSC in the AGM region.
Hematopoietic stem and progenitor cells emerge from the EC of the aorta, by forming ‘hematopoietic clus- ters,8,10,14 including pre-HSC, HSC and HPC. Here, Wip1
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Figure 2. Hematopoietic stem and progenitor cell function was reduced in the embryonic day 10.5-12.5 Wip1-/- aorta-gonad- mesonephros region and yolk sac. (A, B) Colony-forming unit cul- ture assay showed the number of colonies in the aorta-gonad- mesonephros (AGM) (A) and yolk sac (YS) (B) on embryonic day (E)10.5, E11.5 and E12.5 (*P<0.05, **P<0.01, ***P<0.001 for wild-type [WT] vs. Wip1-/-). (C) Direct transplanta- tion assay showed the repopulat- ing ability of E11.5 and E12.5 WT and Wip1-/- AGM. E11.5 (n=4) and E12.5 (n=3): *P=0.03 and ***P<0.001. (D) The lineage out- put of donor-derived granulocyte- macrophage (GM), T and B cells in E12.5 WT and Wip1-/- AGM at 16 weeks after transplantation (n=3, **P=0.0075). (E) Repopulating ability of E11.5 and E12.5 WT and Wip1-/- YS by direct transplantation. (n=3, ***P=0.0004). (F) Donor-derived multilineage output in the periph- eral blood of repopulated recipi- ents of GM, T and B cells repopu- lated by E12.5 YS at 16 weeks after transplantation (n=3, *P=0.017). (G) Repopulating activity of E10.5 WT and Wip1-/- AGM after 3 days of explant cul- ture in vitro (AGMex). *P=0.0237. (H) Donor-derived lineage output of GM, T and B cells repopulated by E10.5 WT and Wip1-/- AGM explants at 16 weeks after trans- plantation. *P=0.0106, ***P<0.001. Each symbol repre- sentes one recipient. Green cir- cles=WT, Red triangles= Wip1-/- . The lines represent average chimerism. CFU-C: colony-forming unit culture; ee: embryo equiva- lent; WT: wild-type; KO: knockout; CFU-Mix: colony-forming unit – granulocyte, erythrocyte, mono- cyte, megakaryocyte; CFU-GM: colony-forming unit – granulocyte- macrophage; BFU-E: burst-form- ing unit – erythroid; GM: Gr- 1+/Mac-1+ cells; T: CD3+ cells; B: B220+ cells.
haematologica | 2021; 106(2)