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E4 enhances human HSPC engraftment in NSG mice
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Figure 5. E2 and E4 enhance human engraftment in immunodeficient male mice. (A) Experimental scheme of transplantation of human hematopoiet- ic stem and progenitor cells (HSPC) into immunodeficient mice. Sublethally irra- diated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were transplanted with human umbilical cord blood CD34+ (CB- CD34+) cells, and 3 days later the ani- mals were treated with vehicle or with daily low doses of either E2 or E4 (2 mg of estrogen per day) for 4 days. Four months after transplantation, human hematopoietic engraftment was evalu- ated in the mouse bone marrow. (B) Percentage of hCD45+ cells in the bone marrow (BM) of male mice transplanted with 5x104 hCB-CD34+ cells 4 months after transplantation. (C) Percentage of hCD34+ cells within the human popula- tion in the BM of male mice transplant- ed with 5x104 hCB-CD34+ cells 4 months after transplantation. (D) Percentage of hCD34+hCD38- within the human population in the BM of the male mice transplanted with 5x104 hCB-CD34+ cells. (E) Percentage of human engraftment (hCD45+) in the BM of secondary NSG mice transplant- ed with 1x106 sorted hCD45+ cells from the primary recipients and analyzed 4 months after transplantation. Data were obtained from four biological repli- cates and are presented by dots and box-plots that represent the interquar- tile range (p75, upper edge; p25, lower edge; p50, midline; p95, line above the box; and p5, line below the box). Statistical significance was analyzed by the Mann-Whitney U test: **P<0.01 and ****P<0.001.
HSPC to promote hematopoietic engraftment directly or indirectly through niche cells. We therefore cultured 5x104 CB-CD34+ cells with 100 nM of E2 or E4 for 4 days and transplanted the resulting cells after culture into NGS mice. As shown in Figure 6A, human engraftment of in vitro estrogen-treated HSPC was lower than that of vehi- cle-treated cells, which might indicate an indirect mecha- nism of estrogens in enhancing hematopoietic engraft- ment in NSG mice. Additionally, there was no difference in the percentage of lymphoid, myeloid or HSPC subpop- ulations among mice in the different groups (Online Supplementary Figure S6A). To gain a better understanding of the difference in engraftment between HSPC treated in vitro with estrogen and the in vivo effect of estrogens after HSC transplantation, we co-cultured 5x104 CB-CD34+ cells with human irradiated BM-MSC in the presence of
nice. As shown in Online Supplementary Figure S5G, human engraftment 4 months after transplantation tended to increase in the female animals treated with either of the two estrogens, although differences between groups were not statistically significant. The percentages of hematopoi- etic progenitors within the human population did not show larger differences between vehicle- and estrogen- treated animals (Online Supplementary Figure S5H). Consequently, there is no clear effect of E2 or E4 on the engraftment of human HSPC in female animals.
E4 affects mesenchymal stromal cells within the mouse hematopoietic niche
To obtain further insight into the positive impact of estrogens on promoting human hematopoietic engraft- ment, we assessed whether estrogens act in vivo on human
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