Page 164 - 2021_06-Haematologica-web

S. Fañanas-Baquero et al.
100 nM of E2 or E4 for 1 week and then transplanted the resulting cells into sublethally irradiated NSG mice. Human engraftment and lineage distribution were similar among mice in the different groups (Figure 6B; Online Supplementary Figure S6B), which indicated that the loss of engraftment ability due to in vitro estrogen-mediated expansion might be offset by the BM-MSC. Next, we examined the contribution of the hematopoietic niche to the engraftment of human HSPC after in vivo estrogen- treatment. To do this, we analyzed the mesenchymal and vascular endothelial compartments of the mouse BM niche 4 months after being transplanted and treated with E2 or E4. The percentages of mouse MSC (mCD140a+, also called Pdgfra+) and vascular endothelial cells (mCD144+, also called VE-Cadherin+) in the non- hematopoietic compartment were analyzed (Online Supplementary Figure S6C). Surprisingly, mCD140a+ cells, but not mCD144+ cells, were increased in the mice treated with E4 in comparison with vehicle-treated animals (Figure 6C and D). To focus on this finding, mice were sublethally irradiated and treated with estrogens without human HSPC transplantation. Surprisingly, there were more nucleated cells in the BM of mice treated with E4 (Online Supplementary Figure S6F). These mouse BM cells were cultured to study their ability to form fibroblast colony-forming units (CFU-F). We identified a tendency to more CFU-F in the BM of estrogen-treated mice than in the BM of vehicle-treated mice (Figure 6E; Online Supplementary Figure S6G), which might indicate a benefi- cial role of estrogens in improving the BM niche after irra- diation.
We then evaluated whether human MSC might interact with these estrogens. So, we analyzed the expression of ESR1 and ESR2 in the human BM-MSC compartment by RT-PCR (Figure 6F) and immunofluorescence (Figure 6G; Online Supplementary Figure S6H). Both estrogen receptors were present in human BM-MSC, indicating that the pres- ence of estrogens could influence the behavior of the human stromal cells and indirectly affect the biology and/or engraftment of humans HSPC. To investigate the effect of estrogens on human BM-MSC, a limiting number of human BM-MSC were seeded and treated with estro- gens and their CFU-F potential was assessed. As shown in Online Supplementary Figure S6I, the estrogens had no effect on human BM-MSC. The numbers of CFU-F dropped when the human BM-MSC had been previously irradiated. However, we observed an increase in the num- ber of CFU-F when the BM-MSC were treated with estro- gens after irradiation (Online Supplementary Figure S6I). In conclusion, estrogens, in particular E4, might facilitate and favor the hematopoietic engraftment of human progeni- tors through enhancing the mesenchymal compartment of the hematopoietic niche, in addition to having a direct effect on HSPC.
Discussion
The present study examines the potential use of estro- gens to modify human HSPC engraftment in BM upon transplantation. On the basis of the differences in the level of human hematopoietic engraftment between female and male recipient mice (Figure 1; Online Supplementary Figure S1), and the expression of estrogen receptors in different subsets of human HSPC (Figure 2; Online Supplementary
Figure S2), we explored the impact of estrogen treatment on hematopoietic cells engraftment. E2 and E4 showed a positive effect on the expansion of these cells in vitro by activating the cell cycle (Figure 3; Online Supplementary Figure S3), with E4 being better tolerated than E2 (Figure 3; Online Supplementary Figure S3). Despite the modest role of these estrogens in modulating human progenitor activity in vitro, we found that E2, and even more E4, was able to boost human hematopoietic engraftment in immunodefi- cient mice (Figure 5; Online Supplementary Figure S5). This better performance of human HSPC in estrogen-treated animals might reflect observed gender differences. Furthermore, an apparent expansion of the mouse mes- enchymal stromal compartment was identified in animals treated with E4, which may suggest an additional indirect regulation of the estrogens, enhancing human hematopoi- etic engraftment through niche regulation (Figure 6; Online Supplementary Figure S6). Thus, estrogens could act directly on HSPC as well as indirectly, through the modification of the BM stroma, or BM niche, to enhance CD34+ cell engraftment. These findings might be clinically relevant, since the use of E4 could facilitate HSPC transplantation when only a limited number of cells can be infused.
We have shown that estrogens improve the engraftment of human cells in immunodeficient mice, which reinforces the role of sex hormones in HSPC regulation and might explain the superior performance of female mice as recip- ients of hematopoietic transplants26 (Figure 1). The impor- tance of estrogens in regulating HSPC functions has been explored for a long time, without any clear conclusion being reached. E3 has been found to trap mouse hematopoietic progenitors in the liver.29 E2 has been described as promoting the proliferation of very primitive mouse HSC. The increase of estrogen levels during preg- nancy has also been associated with greater HSC division, higher HSC frequency and an increase in erythro- poiesis.19,27 E2 has also been reported to expand human CB-CD34+ cells in vitro.28 These data contrast with those previously described by Illing et al., who found that long- term treatment of mice with E2 stimulated murine HSPC in the vascular niche but not in the endosteal niche, impairing long-term reconstitution potential.18 On the other hand, high doses of E2 suppressed hematopoiesis in mouse BM.30 This negative effect caused by estrogens has also been shown by tamoxifen treatment, which increased mouse HSC proliferation, but not self-renewal, and induced apoptosis in short-term HSC and MPP.24 Here, we found that human HSPC had different sensitivities to the four natural estrogens. E1 and, to a lesser extent, E2 and E3 were toxic for human HSPC (Figure 3; Online Supplementary Figure S3). E4 was better tolerated and was able to promote some degree of expansion of the human HSPC by activating their cell cycle and inducing less apop- tosis (Figures 3 and 4; Online Supplementary Figures S3 and S4). These observations might explain the apparently divergent effects described previously for estrogens, since different doses and different estrogens were used in the above-mentioned reports.
We also observed that treatment with E2 or E4 in vivo enhanced human hematopoietic engraftment in male mice transplanted with 5x104 human HSPC (Figure 5B; Online Supplementary Figure S5), but only to a minor degree in ani- mals transplanted with very limited numbers of CB- CD34+ cells (Online Supplementary Figure 5E-G). The appar- ent lack of effectiveness in female mice might be due to
1666
haematologica | 2021; 106(6)