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S. Fañanas-Baquero et al.
numbers, poor homing, low engraftment, or differentiation stress of the HSPC. Different approaches have been attempted to solve these problems, such as using different sources of HSPC,3-5 ex vivo expansion of HSPC6-10 or stimu- lating the HSPC by accessory molecules11,12 or cells.13 However, these approaches require a profound understand- ing of HSPC regulation and how the properties of the cells can be boosted to maximize their efficacy at reconstituting a patient’s blood system after HSPC transplantation.1,14
Estrogen is the primary female sex hormone and, apart from its known role in the reproductive system, it is respon- sible for controlling many cellular and molecular processes, including growth and differentiation. Estrogens act through genomic or nuclear signaling and non-genomic or mem- brane-initiated steroid signaling (MISS), modulating intra- cellular second messengers.15 The four major naturally- occurring estrogens in women are estrone (E1), estradiol (E2), estriol (E3) and estetrol (E4). E1 is the predominant estrogen in postmenopausal women. E2 is considered the active estrogen during the estrous cycle. E3 and E4 are syn- thesized during pregnancy by the placenta and fetal liver, respectively, but their physiological roles are essentially unknown.16
Recent evidence indicates that E2 is involved in regulating the proliferation and lineage commitment of HSC,17 although the studies are few and their results are sometimes contradictory. E2 treatment was able to specifically increase the number of vascular HSC, but the long-term repopulat- ing capacity of the HSC was limited.18 Additionally, this E2 was shown to promote the cell cycle of HSC and multipo- tent progenitors (MPP) and increase erythroid differentia- tion in females, also during pregnancy.19 Furthermore, E2 favors hematopoietic regeneration through the activation of telomerase activity20-22 and the stimulation of the unfolded protein response on mouse HSC, which sustains protein homeostasis to favor hematopoietic regeneration.23 In con- trast, tamoxifen, whose active metabolite (4-hydroxyta- moxifen) acts as an estrogen receptor antagonist, reduces the number of MPP and short-term HSC but activates the proliferation of long-term HSC.24 In addition, E2 might modulate HSC indirectly through activating BM mesenchy- mal stromal cells (MSC). E2 treatment has been described to activate MSC osteogenic differentiation and also pro- motes the secretion of granulocyte-macrophage colony- stimulating factor and interleukin 6, which increased the number of HSC by modulating their niche.25 Therefore, estrogen-mediated regulation of HSPC can also indirectly change the HSC BM niche. For that reason, a full under- standing of the role of estrogens in HSC regulation is essen- tial in order to be able to further develop the clinical poten- tial of these hormones.
Here, we have examined the impact of natural estrogens on human HSPC. Ex vivo, E2 and E4 treatment expanded human HSPC and, more importantly, the administration of E4 to immunodeficient mice previously transplanted with human HSPC enhanced the level of engraftment of human hematopoietic cells.
Methods
Human cord blood-CD34+ cell samples and bone mar- row mesenchymal stromal cells
Umbilical cord blood samples (CB) from healthy donors were provided by the Centro de Transfusión de la Comunidad de
Madrid. All samples were collected with written consent and agreement from the Centro de Transfusión de la Comunidad de Madrid‘s institutional review board (number PKDEFIN [SAF2017-84248-P]). Mononuclear cells were obtained by frac- tionation in Ficoll-hypaque according to the manufacturer’s rec- ommendations (GE Healthcare). Purified CB-CD34+ cells were obtained using a MACS CD34 Micro-Bead kit (Miltenyi Biotec). Cells were frozen in 10% dimethyl sulfoxide solution and stored in liquid nitrogen until their use.
Mononuclear cells from human BM were obtained by Ficoll- Paque Plus density gradient separation from heparinized BM samples obtained from healthy donors after informed consent. All the procedures were in accordance with the Helsinki Declaration of 1975, and its revision in 2000. Samples were cul- tured at 1.6×105 cells/cm2 in MesenCult medium plus supple- ments for human cells (Stemcell Technologies). After 24 h, non- adherent cells were discarded. Fresh medium was added and replaced twice a week. At 80% confluence, adherent cells were trypsinized, washed, and seeded at 4×103 cells/cm2. In all the experiments, BM-MSC were used at passages 5 to 8.
Hematopoietic cell transplant protocol in immunodeficient mice
All the mice were kept under standard pathogen-free condi- tions in the animal facility of CIEMAT. All animal experiments were performed in compliance with European and Spanish leg- islation and institutional guidelines. The protocol was approved by Consejeria de Medio Ambiente y Ordenación del Territorio (protocol number PROEX 078/15).
CB-CD34+ cells were administered through the tail vein of female or male NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice sub- lethally irradiated the day before the transplant with 1.5 Gy. Three days later, the animals were treated with vehicle (olive oil) or daily doses of either E2 or E4 (2 mg of estrogen per day) intraperitoneally for 4 days. Four months after transplantation, the mice were sacrificed and BM was collected from the long bones of these animals. Additionally, when analysis of the hematopoietic niche was involved, the long bones were flushed, cut into small pieces and crushed before being digested with 200 U/mL collagenase IV/2 mg/mL DNaseI in Hanks balanced salt solution at 37°C for 45 min. Human engraftment was analyzed by flow cytometry (LSR Fortessa; BD). The cells were stained with hCD45-APCCy7 and hCD3-APC (BioLegend), hCD45- FITC, hCD33-PE, hCD19-FITC and hCD235a-FITC (Beckman Coulter), hCD34-Pecy5 (Immunotech), hCD38-PE, hCD90- APC, mCD45.1-PE, mCD45.1-Biotin and Ter119-Biotin (BD), mCD140a-APC (Pdgfra-APC, eBiosience) and mCD144-PE (VE- Cadherin-PE, eBiosience). DAPI-positive cells were excluded from the analysis. FlowJo software was used for the analyses.
Additionally, the hCD45+ population from primary mice was sorted in an Influx Cell Sorter (BD) and 1x106 hCD45+ cells were transplanted into sublethally irradiated female secondary NSG recipients. Four months later, the animals were sacrificed and analyzed as previously described.
Results
Engraftment of human cord blood CD34+ cells is favored in female immunodeficient mice
It has been previously described that the engraftment of highly purified human HSC (Lin-CD34+CD38- CD90+CD45RA-) is improved when these cells are trans- planted into immunodeficient female recipients, as com- pared to male recipients.26 To investigate whether this
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haematologica | 2021; 106(6)