Page 266 - 2021_02-Haematologica-web

Letters to the Editor
Wip1 regulates hematopoietic stem cell
development in the mouse embryo
The first adult-repopulating hematopoietic stem cells (HSC) are found in the aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5,1,2 as well as in other sites, such as the embryonic head,3,4 yolk sac (YS) and pla- centa.5-7 Accumulating evidence from ex vivo models sug- gests that HSC emerge from endothelial cells (EC) of the aorta by forming sprouting clusters,8-10 through the pre- HSC I (VE-Cadherin+CD41lowCD45–) and pre-HSC II (VE- Cadherin+CD45+) stages of maturation, and are regulated by various transcription factors and signaling pathways. Less is known about the exact mechanism of maturation of pre-HSC in vivo. Wild-type p53-induced phosphatase (Wip1) is defined as a critical regulator involved in HSC function and lymphoid/neutrophil development.11,12 However, the role of Wip1 in regulating HSC function and pre-HSC maturation in the embryo is still unknown.
Our data have shown that Wip1 is expressed in EC and pre-HSC in the AGM region and fetal liver (FL)/bone marrow HSC,13 suggesting that Wip1 may be involved in hematopoiesis (Online Supplementary Figure S1A). To test whether Wip1 plays a role in hematopoietic development of the embryo, Wip1 homozygous deficient embryos (Wip1-/-, KO) were used in this study. The total cell num- ber was significantly decreased in E12.5 Wip1-/- FL com- pared with wild-type (Wip1+/+, WT) (2.8±0.4×106 vs. 4.1±0.5×106) and E13.5 (5.9±0.5×106 vs. 9.1±0.7×106) (Figure 1A, Online Supplementary Figure 1B). The percent- ages of CD45+ and CD34+c-Kit+ cells were indistinguish- able, but the absolute cell numbers of both populations were obviously reduced, respectively (Figure 1B-E, Online Supplementary Figure S1C, D). To test the hematopoietic progenitor cell (HPC) function, colony-forming unit-cul- ture (CFU-C) assays were performed, and revealed small- er size of burst-forming unit-erythroid (BFU-E), CFU- granulocyte macrophage (CFU-GM) and CFU-granulo- cyte erythrocyte monocyte megakaryocyte (CFU-Mix) colonies in the E12.5 Wip1-/- FL compared with E12.5 WT FL (Online Supplementary Figure S1E). The number of CFU-C per E12.5 Wip1-/- FL was reduced by more than 70% (3.4±0.3×103 E12.5 vs. 14.2±1.4×103), with dramatic reductions in BFU-E, CFU-GM and CFU-Mix numbers. The same trend in reduced CFU-C numbers was observed from E13.5 to E14.5 in Wip1-/- FL. Interestingly, the CFU-C numbers from the same input number of FL cells (2×104 cells) were decreased considerably (Figure 1F, G, Online Supplementary Figure S1F), demonstrating the influence of Wip1 deletion on the potential of HPC in the FL.
Subsequently, flow analysis and transplantation were used to investigate Wip1 function in FL HSC develop- ment. Firstly, there was a significant reduction in the per- centage of HSC of E12.5 Wip1-/- FL compared to WT FL (0.015±0.003% vs. 0.025±0.004%) as revealed by a cock- tail of markers (Lin–CD48–Mac1lowSca-1+CD150+, SLAM HSC). Moreover, the absolute number of SLAM HSC was reduced by more than 30% (419±80 vs. 607±116/embryo equivalent), similar to the decrease in the percentage and absolute number of Lin–Mac1lowSca-1+ cells (including more immature HPC) (Figure 1H, J, Online Supplementary Figure S2A, B). Subsequently, assays following in vivo transplantation by injection of 0.05 embryo equivalent/recipient revealed that six out of 19 recipients given E12.5 Wip1-/- FL cells were repopulated, with 12.7±4.6% chimerism, whereas 15 out of 17 recipients of WT cells achieved chimerism of 65.0±7.8% (Figure 1K,
Online Supplementary Table S1). Multilineage assays showed the reduction of B-cell lineage output (B220+ %) in the peripheral blood from Wip1-/- FL derived-recipients. (Figure 1M, Online Supplementary Figure S2C-G). Secondary transplantation data showed that HSC self- renewing ability was decreased in the Wip1-/- group (Online Supplementary Figure S2H). Although all the recip- ients were repopulated, the average chimerism was decreased in the E14.5 Wip1-/- FL, with reductions in B- and T-cell lineage output and an increase of myeloid cells (Figure 1L, N). Taken together, our findings indicate the involvement of Wip1 in HPC development and HSC activity of embryonic FL with a decrease in lymphoid lin- eage differentiation.
E11.5 embryos with Wip1 deficiency are smaller than their WT littermates (Online Supplementary Figure S3A). The total cell number was decreased from E10.5 to E12.5 AGM (Online Supplementary Figure S3B). Flow analysis data showed that the percentages of CD45 were compa- rable in the Wip1-/- AGM and YS and the corresponding WT tissues; however, the percentage of CD41lowCD45– cells was decreased significantly in the E11.5 AGM region, but not in the YS (Online Supplementary Figure S3C-F), indicating a reduction of HPC. CFU-C assays confirmed that Wip1 ablation leads to a dramatic decrease in HPC function (61±21 vs. 326±49 CFU- C/AGM) at E10.5, including reduced numbers of CFU- Mix and CFU-GM. Similar decreases were found in the E11.5 and E12.5 Wip1-/- AGM (85±13 and 30±8 CFU- C/AGM, respectively) compared with WT AGM (249±46 and 208±39 CFU-C/AGM, respectively) (Figure 2A). High proliferative potential colony-forming cells presenting immature HPC were clearly reduced in the Wip1-/- AGM (Online Supplementary Figure S3G). Meanwhile, similar trends in reduction were found in E10.5-E12.5 Wip1-/- YS (Figure 2B). These results suggest a positive regulatory role of Wip1 in HPC function in the AGM region and YS.
Thereafter, in vivo transplantation assays were per- formed to test the influence of Wip1 on HSC functions. Four of 12 (33%) recipients of E11.5 WT AGM cells showed long-term, high-level multilineage repopulation, but none of nine recipients of Wip1-/- AGM cells were repopulated. Unexpectedly, two of seven recipients of E12.5 Wip1-/- AGM cells were repopulated, with a lower repopulation ratio compared with the WT group (6/6 recipients were repopulated). The B-cell lineage output was diminished in Wip1-/- AGM-derived recipients (Figure 2C, D, Online Supplementary Table S1), consistent with Wip1-/- FL and bone marrow HSC. In E11.5 YS, the ability to engraft was comparable (3/9 with chimerism of 9.1±4.1% vs. 2/6 with chimerism of 15.4±10.5%); how- ever, the engraftment ability was decreased significantly in E12.5 Wip1-/- YS with an increase of myeloid output (8/11 vs. 9/9) (Figure 2E, F). These data, together with the HPC results, indicate that Wip1 affects hematopoietic stem and progenitor cell function mainly in the E11.5 AGM region.
HSC are reported to emerge autonomously at E10.5 (>35 somite pairs) in the AGM region but are inefficient in direct transplantation because of their low activity.1 Explant culture is an efficient tool for HSC expansion/pre-HSC maturation. Explant culture assays revealed a decrease in HSC activity in the recipients receiving E10.5 Wip1-/- AGM (4/9) compared with WT AGM (11/12), with a greater myeloid output at the cost of T lymphoid output (Figure 2G, H), indicating that Wip1 ablation may affect HSC expansion and/or pre- HSC maturation.
We also investigated whether Wip1 influences pre-
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haematologica | 2021; 106(2)