Sex Dependent Impacts of Exogenous Estrogens in Cell Culture Media on HMSC Metabolism and Proliferation

Introduction:: Women are at a higher risk for several musculoskeletal injuries including ACL tears, meniscus tears, scoliosis, osteoarthritis, osteoporosis, and others across their lifespan [1]. Human mesenchymal stromal cells (hMSCs) can be derived from adult bone marrow and adipose tissue and have the capability to differentiate into multiple cell types in vitro and exhibit potent anti-inflammatory and regenerative signaling queues in vivo via the secretome components. HMSCs and their products are especially promising for treatment after musculoskeletal injury where inflammation and a lack of native regeneration make these signaling queues especially necessary. Female cells vary in their exposure and response to cyclic concentrations of estrogens compared to male cells. Phenol red is an indicator of pH commonly utilized in cell culture media that also acts as a non-steroidal estrogen [2]. Fetal bovine serum (FBS) is another ubiquitous cell culture additive that contains varied levels of estrogens, and the source is not controlled or reported across studies [3]. The impact of exogenous estrogens present in standard cell culture media has been investigated in hMSCs in a limited number of studies; however, only one study utilized female MSCs, that were non-human [4]. Herein, we investigated the effects of phenol red, unfiltered FBS, and passage (age) on male and female hMSC metabolism, proliferation, senescence, and differentiation. We hypothesized that exogenous estrogens in the form of phenol red and unfiltered FBS would increase metabolism and proliferation, decrease senescence, and significantly impact osteogenic, chondrogenic, and adipogenic differentiation potential in a sex-based manner.


Materials and Methods:: Bone marrow derived hMSCs were sourced from Rooster Bio. Preliminary data are from a 20 year old female and a 21 year old male, both African American. Studies are underway to increase the donor pool with 4 male donors from 19 – 25 years of age, and 4 female donors from 20 – 29 years of age, matched across ethnicities. Male and female cells were grown in low glucose phenol red (“phenol”, Gibco D6046) or phenol red free (“free”, Gibco D5921) Dulbecco's Modified Eagle's Medium (DMEM) with either normal (“normal”, Atlanta Biological’s 511150) or charcoal dextran filtered (“filtered”, Atlanta Biological’s 511650) FBS. Cells were expanded in E2 deprived, phenol free/filtered media for at least 5 days prior to study. Cells at passages P2, P3, and P4 were cultured for 5 days in media conditions as well as free/filtered + 20 pg/mL 17-β-Estradiol (E2) as a positive control. Prestoblue reagent and Picogreen™ were utilized to assay metabolic activity and cell proliferation, respectively. Cells were fixed for β-Galactosidase for senescence assessment. An optimized proximity ligation assay (PLA) was used to investigate differences in ERα and ERβ homo- and heterodimerization. Following 2 weeks of expansion in media conditions, cells will be cultured for 14-21 days in their respective differentiation media. Following incubation in differentiation media, cells will be stained with alizarin red (osteogenic), Oil Red O (adipogenic), and alcian blue (chondrogenic) to assess impact of media components on trilineage differentiation.


Results, Conclusions, and Discussions:: Phenol red significantly impacted hMSC metabolism at all passages tested (Figure 1. B-E). The phenol/normal condition proliferated significantly higher (P< .05) for female cells across all passages (Figure 1. A). While this agrees with our hypothesis, it is not significantly different from the free/normal group. The free/normal condition had significantly (P< .05) higher metabolism when compared to the phenol/normal condition at passage 4 for both sexes (Figure 1. D-E), and at passage 3 for the female cells. The addition of 20 pg/mL of exogenous human E2 suppressed metabolism and proliferation. Across all passages, female cells were significantly (P< .001) more metabolically active than the male cells in the free/normal condition and the female cells in the phenol/normal condition proliferated significantly more than the male cells. In other studies from our lab using PLA, phenol red significantly impacted estrogen receptor dimerization in fibroblast-like cells and more significantly in female cells. Current investigation is ongoing to assess ER dimerization and impact for downstream transcriptional signaling in male and female hMSCs. This is the first study to investigate hMSC sex differences in culture conditions using both male and female human cells [5]. In contrast to Hong et al., cells in this study did not significantly increase proliferation in response to picomolar concentrations of E2. Instead, E2 significantly decreased both male and female hMSC proliferation and metabolism [5]. Female hMSCs were significantly (P< .001) more metabolically active in the free/normal condition whereas they proliferated significantly (P< .05) more than male cells in the phenol/normal conditions, this suggests activity in these cells that is not proliferative. Effects were most significant (P< .0001) in older (P4) cells, suggesting that perhaps cellular age exacerbates these differences. Estrogen mimetics appear to impact cellular metabolism significantly more than proliferation with this effect more drastic in female cells and higher passage cells motivating the need to investigate these and additional cellular processes including apoptosis. Researchers should take care when designing future hMSC studies to include female samples and consider not utilizing exogenous estrogens in their media or consider the effects to downstream behavior and function.


Acknowledgements (Optional): :

References (Optional): : [1] D. A. Hart, “Sex differences in musculoskeletal injury and disease risks across the lifespan: Are there unique subsets of females at higher risk than males for these conditions at distinct stages of the life cycle?,” Front. Physiol., vol. 14, p. 1127689, Apr. 2023, doi: 10.3389/fphys.2023.1127689.

[2] W. V. Welshons, M. F. Wolf, C. S. Murphy, and V. C. Jordan, “Estrogenic activity of phenol red,” Molecular and Cellular Endocrinology, vol. 57, no. 3, pp. 169–178, Jun. 1988, doi: 10.1016/0303-7207(88)90072-X.

[3] M. J. Sikora, M. D. Johnson, A. V. Lee, and S. Oesterreich, “Endocrine Response Phenotypes Are Altered by Charcoal-Stripped Serum Variability,” Endocrinology, vol. 157, no. 10, pp. 3760–3766, Oct. 2016, doi: 10.1210/en.2016-1297.

[4] L. Hong, H. Sultana, K. Paulius, and G. Zhang, “Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: A gender difference,” The Journal of Steroid Biochemistry and Molecular Biology, vol. 114, no. 3–5, pp. 180–185, Apr. 2009, doi: 10.1016/j.jsbmb.2009.02.001.