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S. Fañanas-Baquero et al.
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Figure 2. Human hematopoietic stem and pro- genitor cells express estrogen receptors. (A) Representative immunofluorescent image of umbilical cord blood CD34+ (CB-CD34+) cells stained with anti-ESR1 (green), anti-hCD34 (red) and DAPI (blue). The insert, showing ESR1+ CD34+ cells (marked with arrows), is an enlargement of the white boxed area. (B) Representative immuno- fluorescent image of CB-CD34+ cells stained with anti-ESR2 (green), anti-hCD34 (red) and DAPI (blue). The insert, showing ESR2+ CD34+ cells (marked with arrows), is an enlargement of the white boxed area. (C) Qualitative real-time poly- merase chain reaction (qRT-PCR) analysis of ESR1 expression of sorted hematopoietic stem cells/multipotent rogenitors (HSC/MPP: hCD34+hCD38-hCD45RA-), multilymphoid progeni- tors (MLP: hCD34+hCD38-hCD45RA+) and commit- ted hematopoietic progenitors (Hem Prog: hCD34+hCD38+). (D) qRT-PCR analysis of ESR2 expression of sorted HSC/MPP, MLP and hematopoietic progenitors. Data were obtained from three biological replicates and are presented as the mean ± standard deviation. Statistical sig- nificance was analyzed by one-way analysis of variance with the Fisher least significant differ- ence test: no significant differences were found.
after 4 days of culture in the presence of 100 nM E2 or E4. Estrogens, particularly E4, induced an increment of cells in G2/M phase (Figure 3C; Online Supplementary Figure S3H), which might explain the tendency of these two estrogens to expand human hematopoietic progenitors.
Previously, E2 was described to have a positive role in enhancing both CB-CD34+ cell proliferation and in vitro hematopoietic progenitor potential after more than week of in vitro treatment.28 Hence, we cultured human HSPC in the presence of the lowest and best tolerated doses of E2 or E4 for 8 days. We detected a significant expansion of human progenitors with E4 at all the concentrations tested (Figure 3D). A similar effect was identified with 100 nM E2. The better tolerance of E4 over E2 was confirmed, since all the tested concentrations of E4 were non-toxic to CB-CD34+ cells (Figure 3D). The in vitro functionality of the estrogen-treated HSPC was assessed with colony- forming unit (CFU) assays. We did not observe any differ- ences among the groups in CFU numbers or CFU types (Online Supplementary Figure S3I).
In order to assess which estrogen receptor was involved in the effect of these molecules in human HSPC, the cell cycle of CB-CD34+ was determined in the presence of these two estrogens together with either ESR1 antagonist (MPP), ESR2 antagonist (PHTPP) or GPER1 antagonist (G-
tically at any of the concentrations used. On the other hand, the lowest concentrations of E2 and E4 promoted the expansion of these cells, but at high doses they impaired cell growth. A similar behavior was detected when different subpopulations of hCD34+ cells were ana- lyzed (Figure 3B; Online Supplementary Figure S3A-E). E1 prevented the expansion of hCD34+hCD38- cells (Online Supplementary Figure S3C), MLP (Online Supplementary Figure S3D), MPP (hCD34+hCD38-hCD90-hCD45RA-) (Online Supplementary Figure S3E) and most primitive HSC (hCD34+hCD38-hCD90+hCD45RA-) (Figure 3B). The data for the rest of the tested estrogens showed an apparent amplification of these primitive populations when low concentrations of the hormones were used, but at the highest concentrations, they were toxic (Figure 3B; Online Supplementary Figure S3C-E). It is important to highlight that the best tolerated estrogen was E4. Concentrations up to 10 mM of E4 seemed not to be detrimental to any of these HSPC subsets, including primitive HSC. In contrast, E2 induced apoptosis of HSPC at high doses (Online Supplementary Figure S3F and G), as previously described for this estrogen and tamoxifen.24,27 However, only human HSPC cultured in the presence of the highest concentra- tion of E4 showed some induction of apoptosis. Furthermore, we analyzed the cell cycle of CB-CD34+ cells
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haematologica | 2021; 106(6)