Abstract
Sex-specific cell culture methods and microphysiological systems can enhance our understanding of how biological sex influences health and disease. Here, we investigated the effects of estradiol and dihydrotestosterone on primary human lung and ocular fibroblasts as well as in human umbilical vein and retinal microvascular endothelial cells from both female and male donors. Treatment of female cells with estradiol and male cells with dihydrotestosterone in 2D culture significantly enhanced proliferation, mitochondrial membrane potential, and upregulated genes associated with bioenergetics and stress responses. Conversely, treatment of female cells with dihydrotestosterone and of male cells with estradiol decreased bioenergetic potential and inhibited cell proliferation. A microphysiological model of bulk tissue vasculogenesis revealed that estradiol enhances vasculogenesis in female tissues and inhibits vasculogenesis in male tissues. Collectively, these findings demonstrate that the sex hormone composition of culture medium significantly influences bioassay readouts in a sex-specific manner.
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Introduction
Biological sex is a genetic modifier that influences normal physiology and the incidence, progression, and treatment responses of diseases including cancers, cardiometabolic disease, and organ fibrosis1,2,3,4,5. Animal models allow for comparison of both sexes, but deviations from human physiology reduce the accuracy of preclinical studies6,7,8. Human cell culture models and bioengineered technologies such as microphysiological systems (MPS) have largely failed to account for sex differences due to a lack of sex-based culture methods9,10. The percentage of publications reporting cell sex has increased significantly in recent years, but most studies ignore the role of sex hormones11,12,13. While sex chromosomes produces cell autonomous genetic sex differences14, defining the interplay of chromosomal sex and sex hormones, such as estradiol (E2) and dihydrotestosterone (DHT), is critical to understand the expression of sex-specific cellular phenotypes15,16,17,18,19,20.
E2, the main estrogen in reproductive aged females, is involved in reproduction and metabolic homeostasis21,22. Likewise, DHT, a potent androgen derived from testosterone, is essential for male secondary sexual characteristics and male reproductive function23,24. Both E2 and DHT influence metabolic processes in multiple organ systems, including bone mineral density, muscle mass and insulin sensitivity, brain and immune function, and cardiovascular health21,24,25. Sex hormone levels fluctuate considerably over the lifespan of females and males. Post-puberty and pre-menopause, female E2 levels fluctuate throughout the menstrual cycle from approximately 20–350 pg/ml (0.073–1.28 nM) in the early to mid-follicular phase to 150–750 pg/ml (0.55–2.75 nM) in the midcycle peak. After menopause, female E2 levels drop to less than 20 pg/ml (<0.073 nM). Male E2 levels are more stable and remain between 10 and 50 pg/ml (0.037–0.18 nM)26. Male DHT levels range from 110 to 950 ng/ml (0.47–2.65 nM) with steady age-related decline of androgen levels after 30 years of age. Premenopausal female DHT levels average 90 ng/ml DHT (0.3 nM), decreasing to an average of 30 ng/ml (0.1 nM) after menopause24. Notably, the loss of estrogens and androgens in men and women via natural aging or disease is believed to contribute to a wide range of pathologies2,22. Thus, developing in vitro models that recapitulate sex-specific physiological differences will require optimization of sex hormone concentrations in the culture medium in accordance with the sex of the cells.
In this study, we report the sex-specific effects of E2 and DHT on primary cells in 2D culture and in 3D bulk tissue vasculogenesis models using multiple types of primary human endothelial cells and fibroblasts. Our data shows that treating female cells with E2 and male cells with DHT significantly increases cellular bioenergetics and proliferation, whereas treatment of female cells with DHT and male cells with E2 conversely exerts inhibitory effects in all cell types tested. Similar sex-specific hormone effects were measured in a 3D tissue vasculogenesis assay. These findings demonstrate the importance of developing sex-based methods for standard cell culture systems and tissue engineered models to improve translational relevance and accelerate the discovery of sex-based precision therapies.
Methods
Experimental design
For 2D immunofluorescence assays and RT-qPCR studies, 4 donors per sex were used for HUVEC and HLF studies while 3 donors per sex were used for HRMVEC and HOF. All donors were utilized individually without pooling. In vasculogenesis assays, HLF and HUVEC were randomly assigned into sex-matched pairs, and 3 pairs were used. See individual experiment sections for replication information. See Supplemental Fig. 1 for cell donor information. All cells were sexed via RT-qPCR for SRY gene to confirm sex was as listed by supplier, and all cells were analyzed via RT-qPCR to ensure expression of sex hormone receptors (Figure S1). Fibroblasts were grown in FibroLife fibroblast complete media kit (Lifeline Cell Technologies, LL-0011) and endothelial cells were grown in VascuLife VEGF endothelial medium complete kit (Lifeline Cell Technologies, LL-0003). Charcoal-stripped hormone depleted media was formulated using the same base Lifeline kits with phenol red and fetal bovine serum (FBS) omitted. Instead, 2% charcoal stripped serum (Gibco, 12676029, Lots #76-029, 74-229) was added. Sex hormones were dissolved in in molecular grade ethanol (Sigma Aldrich, E7023).
2D cell culture
Primary human lung fibroblasts (HLF), human ocular fibroblasts (HOF), human umbilical vein endothelial cells (HUVEC), and human retinal microvascular endothelial cells (HRMVEC) from both XX and XY donors were sourced for use in these studies (ages 20–45). HLF and HOF were expanded in FibroLife fibroblast complete media kit (Lifeline Cell Technologies, LL-0011) and HUVEC were grown in VascuLife VEGF endothelial medium complete kit (Lifeline Cell Technologies, LL-0003). Cells were expanded until passage 3, and cells from passages 3 through 7 were used in all experiments.
Ki67 immunofluorescence
XX and XY HUVEC, HRMVEC, HOF and HLF were seeded at a density of 10,000 cells per well in 24-well tissue culture plates and grown in cell type-specific Lifeline expansion medium as outlined above for 48 hours. After 48 hours, cells were switched to the same media formulated to be phenol red-free and supplemented with 2% charcoal stripped serum for 24 h to ensure complete hormone starvation. Following hormone starvation, cells were exposed to media supplemented with 0.1 nM, 1 nM, or 10 nM E2 or 0.01 nM, 0.1 nM, 1 nM or 10 nM DHT dissolved in ethanol for 48 h. Ethanol vehicle was added to the medium for all hormone-free controls. 48 h was selected for the duration of hormone exposure based on the results of RT-qPCR assays that showed significant gene expression changes at 48 h while minimizing the influence of trophic effects at longer timepoints. After 48 h of culture, monolayers were fixed with 4% paraformaldehyde at room temperature for 15 min. Monolayers were then gently washed with Dulbecco’s Phosphate Buffered Saline (PBS, Gibco, 14190144) 3 times. Fixed well plates were stored covered in PBS at 4 °C until staining. Samples were blocked and permeabilized with 1% bovine serum albumin (BSA, Sigma Aldrich, A4737) and 0.1% Triton-X (Sigma Aldrich, X100) in PBS at room temperature for 20 min. Rabbit anti-Ki67 antibody (Abcam, ab16667, Lot # 1090780-56) was added at 10 μL/mL in 0.1% BSA in PBS. Plates were rocked for 2 hours protected from light at a gentle rock and then washed with PBS 4 times. Secondary antibody conjugated with Alexa Fluor 594 (Abcam, ab150092) at 4 μL/mL, 4 μL/mL 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, H21486) to label nuclei, and 4 μL/mL Alexa Fluor 488 Phalloidin (Thermo Fisher, A12379) to label actin were added in 0.1% BSA in PBS for 40 min in a dark, gentle rocking. Plates were then washed again in PBS and stored covered in PBS at 4 °C protected from light until imaging. 4 donors per sex were used for HLF and HUVEC, and 3 donors per sex were used for HOF and HRMVEC. 3 wells per condition per donor were utilized. 3 images per well were analyzed.
RT-qPCR
Cells were plated at 5000 cells per well in 6 well plates. At 50% confluence, cells were changed to phenol red-free media supplemented with charcoal stripped serum for a 24 h hormone starvation. After 48 h of starvation, we exposed cells to 0.1 nM, 1 nM or 10 nM E2 or 0.01 nM, 0.1 nM, 1 nM, and 10 nM DHT. E2 and DHT were dissolved in ethanol, and control groups were supplemented with the same volume of ethanol as a vehicle control. E2 and DHT were replenished every 24 h, and media was changed every 48 h. Cells were collected via trypsinization at 48 h, 72 h, and 168 h of exposure. If not isolated immediately, cell pellets were stored in RNAlater (Thermo Fisher) until RNA isolation. mRNA was isolated from collected samples via Qiagen RNEasy kit following the manufacturer instructions, and sample purity was validated by measurement of the 260/280 nM and 260/230 nM ratios using a Nanodrop spectrophotometer (Thermo Fisher). mRNA samples were stored at −80 °C for long term storage. cDNA was synthesized using qScript cDNA SuperMix (QuantaBio) according to the supplier recommended protocol, diluted 1:10 in UltraPure DNase and RNase free water (Thermo Fisher), and stored at −20 °C. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the expression of genes associated with energetics and stress response in all cells. RT-qPCR was completed using SybrGreen PowerUp on the ThermoFisher StepOnePlus system (Thermo Fisher). Primers used are listed in Table 1. GAPDH was utilized as the housekeeping gene. RT-qPCR results were analyzed via the 2−ΔΔCt method. 4 donors per sex were used for HLF and HUVEC, and 3 donors per sex were used for HRMVEC and HOF. 3 biological replicas per donor and 3 technical replicas per gene were analyzed.
Tetramethylrhodamine methyl ester (TMRM) mitochondrial membrane potential assay
Monolayers were cultured as described in Ki67 immunofluorescence. After 48 h of hormone culture, cells were exposed to 100 nM tetramethylrhodamine methyl ester (TMRM) dissolved in DMSO (Sigma Aldrich, D8418) for 30 min at 37 °C per manufacturer instructions (Thermo Fisher, T668). Monolayers were washed thoroughly with PBS and imaged immediately. 4 donors per sex were used for HLF and HUVEC, and 3 donors per sex were used for HOF and HRMVEC. For each end condition, 3 wells were analyzed for each time point using 3 images per well.
Fabrication of PDMS microarrays
A 9-well milliscale tissue array was designed in SolidWorks (Dassault Systèmes) and transferred into PreForm 3D Printing Software (FormLabs). Molds were printed using a Form3 SLA 3D printer (FormLabs). After printing the molds were washed, cured, and flattened to generate acceptable molds for polydimethylsiloxane (PDMS) soft lithography27. As PDMS is known to absorb small, hydrophobic molecules such as steroid hormones28, molds were polyurethane coated to increase smoothness of PDMS and reduce surface area to minimize absorption as we have previously reported27. Absorption of sex hormones was further mitigated by cross-mixing a polyethylene glycol (PEG) and PDMS self-segregating polymer (PDMS-PEG) into the PDMS mixture prior to PDMS curing29. The ideal amount of PDMS-PEG for our purposes was found to be 0.25% by weight as this endowed the material with the surface properties we desired while maintaining the benefits of traditional PDMS. As such, PDMS was mixed at a 1:10 ratio of elastomer to fixative agent (Sylgard 184, Ellsworth Adhesives). The PDMS-PEG copolymer was then added to a total of 0.25% weight (Gelest, DBE-712). The polymer was then mixed thoroughly and degassed. PDMS was poured into molds, degassed again, and kept in an oven at 60 °C for at least 4 hours to ensure complete curing. Prior to use, we sanitized devices via UV light sterilization for 2 hours. Poor protein adsorption and cell seeding on untreated PDMS surfaces is well-documented30,31,32. Thus, surfaces of the array wells were treated with a 5 mg/ml polydopamine (PDA) solution for 2 h in UV light to facilitate stable anchorage of ECM hydrogels33. Devices were then stored in a dark space at room temperature until use.
Nile Red assay
Nile Red absorption assays were completed to test that PDMS-PEG copolymer microarrays would prevent hormone absorption. Nile Red, a fluorescent stain with similar size to E2, has been used in previous studies to assay PDMS absorption of small molecules28. 1 μg/mL Nile Red (Thermo Fisher) was dissolved in ethanol and loaded into central wells of small PDMS copolymer devices and allowed to sit for 1 h. Devices were washed with PBS 3 times. PDMS with or without copolymer made on coated and non-coated molds were imaged under fluorescence. Images were quantified using the average gray intensity across the well as calculated in FiJi.
3D vasculogenesis assay
HUVEC and HLF were expanded using the same techniques used in 2D culture. Polydopamine (PDA)-treated PDMS microarrays were loaded with 2 × 106 cells/ml HLF and 2 × 106 cells/ml HUVEC in a hydrogel comprised of blended collagen type I (2.25 mg/ml collagen type I, Corning) and fibrin (5 mg/mL fibrinogen activated with 1 U/ml thrombin, Sigma Aldrich). This hydrogel composition was selected to combine the vascularization promoting properties of fibrin with the suprastructural stability provided by collagen gels34,35,36. Hydrogels were allowed to solidify at 37 °C for 30 min. Stromal vascular tissues were cultured in vascular cell growth media supplemented with VEGF (ATCC), 25 μg/mL aprotinin (Sigma Aldrich), and 2% FBS for 96 h. Media was changed to phenol red free media containing 2% charcoal stripped serum (Gibco) for complete hormone starvation for 24 h before exposing the gels to 0 nM, 1 nM, or 10 nM E2 in ethanol or 0 nM, 1 nM, or 10 nM DHT for 4 days. For each sex, 3 donor combinations were utilized for 3 gels per condition and 3 images per gel. See Supplemental Fig. 7H for workflow.
Immunofluorescence and 3D whole mount staining
3D tissues from the PDMS tissue arrays were processed for whole mount staining. Tissues were fixed in 4% paraformaldehyde (Sigma Aldrich) for 1 h at room temperature and then overnight at 4 °C. Tissues were then washed with several changes of PBS and stored in PBS at 4 °C until stained. Gels were stained using 4 μL/mL (DAPI) to label nuclei, 4 μL/mL Alexa Fluor 488-conjugated Phalloidin (Thermo Fisher, A12379) to label F-actin, and 20 μL/mL Ulex Europeas agglutinin I (UEA-1, Vector laboratories, RL-1062-2) to specifically label endothelial cell lectins. Stains were prepared in PBS with 0.2% Triton-X and 1% BSA (Sigma Aldrich). Devices were loaded with staining cocktail, ensuring complete coverage of the gels, and rocked gently for 1 h at room temperature. Gels were then refrigerated overnight, rocked for an additional hour the next day at room temperature, washed in PBS, and stored covered with PBS at 4 °C until imaging.
Imaging and image analysis
All 2D and 3D samples were imaged on an inverted Nikon C2 laser scanning confocal microscope (LCSM) equipped with a Nikon DS-FI3 camera. Samples from a given staining cohort were imaged at a fixed laser intensity and exposure time. For 2D cultures, 3 images of each sample were taken at randomized points in the central regions of the well to avoid edge effects when imaging. Analysis of 2D stains was performed in FiJi. TMRM images were converted to grayscale and segmented to the area of the cells utilizing an overlayed DIC image of the sample. Mean fluorescence intensity of the region of the cells was measured, and the average intensity across the image was recorded. This process was completed for each of the 3 images taken from each well. The average of these 3 measurements was then used as the average intensity across the entire well. Ki67 staining was analyzed in FiJi, using the DAPI co-stain as the mask for regions of interest (cell nuclei). The threshold intensity of DAPI images was adjusted to the sharply capture cell nuclei and turned into a binary mask which was overlaid on the Ki67 channel. Ki67 positive cells were counted via particle analysis.
Each tissue from the vasculogenesis assay was imaged with 3 full thickness Z-stacks, excluding the excluding a rim of tissue at the interface with PDMS surfaces. 3D image datasets were analyzed via MATLAB (Mathworks, R2021b). Max intensity projections of the Z-stacks were saved at TIFF files and exported to MATLAB for filtering and quantification. Vascular images were smoothed with a gaussian filter, low intensity noise was filtered out, and images were then denoised via a pretrained neural network27,37. The open-source segmentation tool REAVER was utilized to segment vascular networks and quantify morphometric parameters37. For non-participating cells, TRITC channels were isolated and analyzed for particles with high roundness. Image analysis results were exported to GraphPad Prism V 9.2 for statistical analysis.
Statistics and reproducibility
All statistics were completed in GraphPad Prism V 9.2. For analysis of TMRM and Ki67 indexes, statistical analysis was completed via two-way ANOVA between sex and hormonal conditions with a 95% confidence interval (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). Vasculogenesis data was compared via two-way ANOVA comparing between sex and hormone conditions. Data was assessed and found to be normally distributed per a Shapiro-Wilk test. Variances were homogeneous per Levene’s test for homogeneity of variances. All samples used to collect datasets were independent, thereby meeting the necessary assumptions for two-way ANOVA. The number of donors per cell type per sex and the number of technical replicates per experiment are included in previous Methods sections for each assay and in the corresponding Figure captions.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Results
Sex-specific hormone effects on fibroblast and endothelial cell proliferation
Female (XX) and male (XY) fibroblasts and endothelial cells were treated with escalating doses of E2 and DHT. Human lung fibroblasts (HLF) and human umbilical vein endothelial cells (HUVEC) were included in the study due to their widespread use in vascularized MPS platforms and cell biology research27,38,39. Human ocular fibroblasts (HOF) and human retinal microvascular endothelial cells (HRMVEC) were included to explore the potential impacts of known tissue-specific responses to external stimuli in fibroblasts and endothelial cells40,41,42,43,44,45. Accordingly, dose-dependent effects of E2 and DHT were tested in HLF, HOF, human dermal fibroblasts (HDF), HUVEC, and HRMVEC. E2 was administered at 0.1 nM, 1 nM, and 10 nM and DHT was administered at 0.01 nM, 0.1 nM, 1 nM, and 10 nM. These dosage ranges were chosen to account for the average ranges of endogenous hormones22. Cells were cultured for 48 h and then stained for the cell cycle marker Ki67 to measure the effects of E2 and DHT on proliferation46. As proliferation is central to many biological processes including tissue maintenance and embryogenesis, and sufficient cell proliferation is required to ensure adequate cell populations for biotechnology applications, we opted to measure cell proliferation along with gene expression and bioenergetic capacity in 2D culture47,48.
The baseline percentage of Ki67 positive cells of HLF in hormone-free conditions were 54 + /− 6% in XY HLF and 76.5 + /− 5.5% in XX HLF (Fig. 1A–C). DHT significantly increased the percentage of Ki67 positive XY HLF in a dose-dependent manner, reaching 92.5 + /− 5% at 10 nM DHT. E2 significantly increased the XY HLF Ki67 index at 0.1 nM (74 + /− 5%), but 1 nM and 10 nM had no significant effect (Fig. 1B). DHT had no significant effect on Ki67 positivity of XX HLF at 0.01 nM and 0.1 nM, but significantly decreased the percentage of Ki67 positive cells at 1 nM (65 + /− 2.5%) and 10 nM (60 + /− 6%). E2 significantly increased the percentage of cells positive for Ki67 in XX HLF in a dose-dependent manner, reaching 92.5 + /− 2% at 10 nM E2 (Fig. 1C). Sex-specific effects of E2 and DHT on proliferation measured in HLF were largely conserved in ocular fibroblasts (HOF) (Fig. 1D–F). The percent of cells positive for Ki67 indexes in HOF in hormone-free baseline culture were 58 + /− 3% for XY cells and 71 + /− 5% for XX HOF. DHT significantly increased the Ki67 positive percentage of XY HOF, peaking at 85 + /− 2.5% at 1 nM (Fig. 1E). E2 significantly increased the percentage of Ki67 positive cells in XY HOF at 0.1 nM, but higher doses of 1 and 10 nM decreased the percentage of Ki67 cells (Fig. 1E). DHT again significantly decreased the percentage of Ki67 positive female HOF at 1 nM (57.5 + /− 6.5%) and 10 nM DHT (58 + /− 8.5%). E2 increased the percentage of Ki67 positive XX HOF in a dose-dependent manner, but the effect was only significant at 10 nM, reaching 84 + /− 2% (Fig. 1F). Additional analysis of HLF in “standard” media containing 2% FBS showed non-significant differences compared to the 1 nM E2 group, in line with the average E2 content of FBS (Figure S2)49.
A Representative micrographs of XY (top row) and XX (bottom row) HLF exposed to varying concentrations of DHT and E2 for 48 h. Stained with DAPI (blue), F-actin (green), and cell cycle marker Ki67 (red). Scale bar = 200 µm. B Percentage of Ki67-positive XY human lung fibroblasts (HLF) after 48 hours of culture with DHT doses of 0, 0.01, 0.1, 1, and 10 nM, and E2 doses of 0, 0.1, 1, and 10 nM. The same dose responses are shown for all other Ki67-positivity plots. C Percentage of Ki67-positive XX HLF after 48 hours of culture with the indicated doses of E2 and DHT. D Representative micrographs of XY (top row) and XX (bottom row) human ocular fibroblasts (HOF) exposed to varying concentrations of DHT and E2 for 48 h. Stained with DAPI (blue), F-actin (green), and cell cycle marker Ki67 (red). Scale bar = 200 µm. E Percentage of Ki67-positive XY HOF after 48 h of culture with the indicated doses of E2 and DHT. F Percentage of Ki67-positive XX HOF after 48 h of culture with the indicated doses of E2 and DHT. All samples compared against vehicle control groups via two-way ANOVA. n = 4 donors per sex for HLF, 3 donors per sex for HOF, 3 monolayers per condition per donor, 3 images per monolayer. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Bars represent SEM. Dots represent per donor average.
Sex-specific effects of E2 and DHT on endothelial cell proliferation followed the same general trends measured in fibroblasts. Baseline hormone-free Ki67-positive percentages were 75.5 + /− 3% in XY HUVEC and 71 + /− 5% in XX HUVEC (Fig. 2A-C). DHT significantly increased the percentage of Ki67 positive XY HUVEC at 1 nM (89 + /− 2%) and 10 nM (92.5 + /− 5%). E2 significantly decreased the percentage of Ki67 positive cells at 1 nM (56 + /−4.5%) and 10 nM (42 + /− 3.5%). In contrast to the increase measured in XY fibroblasts (Fig. 1B, E), 0.1 nM E2 did not increase the Ki67 positive percentage of XY HUVEC (Fig. 2B), suggesting potential lineage-specific concentration dependence. DHT decreased the percentage of Ki67 positive XX HUVEC, reaching significance at 10 nM (57 + /− 4%). E2 again increased the percentage of Ki67 positive XX HUVEC in dose-dependent manner, reaching significance at both 1 nM (87 + /− 3.5%) and 10 nM (90 + /−3%) (Fig. 2C). Again, as in HLF, HUVEC at 1 nM demonstrated non-significant differences compared to cells grown in FBS (Figure S3). HRMVEC responses to sex hormone stimulation mirrored HUVEC. We noted that HRMVEC, XX cells in particular, had a slightly more elongated spindle-like shape compared to the cobblestone appearance of endothelial cells from larger vessels, such as HUVEC (Fig. 2D); this is in line with other reports from the literature that microvascular cells may exhibit this different shape until confluence50,51. In vehicle controls, the baseline Ki67 positivity percentage was 76 + /− 3.5% for XX HMRVEC and 73.5 + /− 8% for XY HRMVEC (Fig. 2D–F). DHT significantly increased the percent of Ki67 positive XY HMRVEC to 88 + /− 4% at 1 nM and 81.5 + /− 4% at 10 nM. As measured with HUVEC, all levels of E2 exposure decreased the percentage of cells positive for Ki67, reaching significance at 1 nM (64 + /− 9.5%) and 10 nM (62 + /− 3%) (Fig. 2E). Conversely, DHT caused a small non-significant increase in XX HRMVEC Ki67 positivity at 0.01 nM (77 + /− 2.5%) and all higher doses significantly decreased the percentage of Ki67 positive cells (65.5 + /− 2.5% at 0.1 nM, 52.5 + /− 4% at 1 nM, and 39 + /− 3.5% at 10 nM). Escalating doses of E2 induced a non-significant trend of increased XX HRMVEC Ki67 positivity (Fig. 2F).
A Representative micrographs of XY (top row) and XX (bottom row) human umbilical vein endothelial cells (HUVEC) exposed to varying concentrations of DHT and E2 for 48 h. Stained with DAPI (blue), F-actin (green), and cell cycle marker Ki67 (red). Scale bar = 200 µm. B Percentage of Ki67-positive XY HUVEC after 48 h of culture with DHT doses of 0, 0.01, 0.1, 1, and 10 nM, and E2 doses of 0, 0.1, 1, and 10 nM. The same dose responses are shown for all other Ki67-positivity plots. C Percentage of Ki67-positive XX HUVEC after 48 hours of culture with the indicated doses of E2 and DHT. D Representative micrographs of XY (top row) and XX (bottom row) human retinal microvascular endothelial cells (HRMVEC) exposed to varying concentrations of DHT and E2 for 48 h. Stained with DAPI (blue), F-actin (green), and cell cycle marker Ki67 (red). Scale bar = 200 µm. E Percentage of Ki67-positive XY HRMVEC after 48 h of culture with the indicated doses of E2 and DHT. F Percentage of Ki67-positive XX HRMVEC after 48 h of culture with the indicated doses of E2 and DHT. All samples compared against vehicle control groups via two-way ANOVA. n = 4 donors per sex for HUVEC, 3 donors per sex for HRMVEC, 3 monolayers per condition per donor, 3 images per monolayer). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Bars represent SEM. Dots represent per donor average.
Sex-specific hormone effects on fibroblast and endothelial cell gene expression
Cell proliferation increases energy demand and is often accompanied by increased ATP generation52. Additionally, higher rates of proliferation require increased protein production which can potentially induce endoplasmic reticulum (ER) stress without proper regulation53. Accordingly, as sex hormone stimulation exerted significant effects on cell proliferation in culture, we measured changes in the expression of genes associated with bioenergetics and proteostasis under the same culture conditions. Specifically, we measured mRNA levels of the Na + /K+ ATPase (ATP1A1) which is responsible for ion pumping driven by ATP consumption, somatic cytochrome C (CYCS) which encodes a central component of the mitochondrial electron transport chain, and heat shock protein-60 (HSPD1) which acts as a chaperone during ER stress54,55,56,57. XX and XY HLF, HOF, HUVEC, and HRMVEC were exposed to 0.01 nM–10 nM DHT and 0.1 nM-10 nM E2, with the addition of 48 h=, 72 h, and 168 h (7 days) time points to explore potential adaptations after prolonged hormone exposure.
All doses of DHT induced upregulation of ATP1A1, CYCS, and HSPD1 expression in XY HLF that persisted for 7 days. Escalating doses of E2 decreased expression of these genes, although the lowest dose of 0.1 nM E2 slightly increased expression at 48 and 72 h. E2-induced changes in gene expression returned to near baseline levels by 7 days at 1 nM and 10 nM E2, suggesting a more transient effect (Fig. 3Aleft). XX HLF showed opposite trends, with a transient upregulation of all 3 genes at 0.01 nM DHT, while higher doses of 1 and 10 nM induced downregulation. E2 induced upregulation of all 3 genes at all time points and doses in XX HLF, again returning to near baseline levels by 7 days (Fig. 3Aright). Sex-specific effects of E2 and DHT on expression of these genes in HLF was largely conserved in ocular fibroblasts (HOF). XY HOF upregulated HSPD1, ATP1A1, and CYCS in response to DHT for up to 7 days. XY HOF showed a slight, transient upregulation of HSPD1 and CYCS in response to 0.1 nM E2, while 1 nM and 10 nM E2 induced transient downregulation of HSPD1, ATP1A1, and CYCS (Fig. 3Bleft). XX HOF exhibited an upregulation of HSPD1 and ATP1A1 in 0.01 nM DHT at 48 hours and 72 hours. Interestingly, 0.01 nM and 0.1 nM DHT both induced an increase in CYCS at all time points, but 1 and 10 nM DHT induced downregulation of all genes that trended toward baseline levels by 7 days. E2 upregulated of all 3 genes at all time points in XX HOF (Fig. 3Bright). Similar gene expression responses were measured when human dermal fibroblasts (HDF) were exposed to E2 (Figure S4).
A, B RT-qPCR results for energetic and stress-related genes in HLF (A) and HOF (B) exposed to the indicated doses of DHT and E2 over 7 days with baseline expression (Fold change = 1) represented in white, upregulation (Fold change > 1) shown in red, and downregulation (Fold change <1) shown in blue. C, D Similar results are seen in the same genes in human umbilical vein endothelial cells (HUVEC) (C) and HRMVEC (D). Analyzed using the 2−ΔΔCt method with GAPDH as a housekeeping gene and the same cells grown in media containing charcoal stripped serum as a control. n = 4 donors per sex for HLF and HUVEC, 3 donors per sex for HOF and HRMVEC, 3 technical replicas per donor, 3 replicates per gene.
Sex-specific endothelial cell gene expression responses to sex hormone stimulation followed similar trends measured in fibroblasts (Fig. 3C, D). DHT exposure induced upregulation of all 3 genes in XY HUVEC, but expression levels of ATP1A1 and CYCS again returned to near baseline by 7 days. Escalating doses of E2 induced downregulation of all 3 genes in XY HUVEC, with a transient upregulation at the lowest dose of 0.1 nM (Fig. 3Cleft). In XX HUVEC, 0.01 nM DHT induced slight upregulation that returned to baseline by 7 days, while 1 and 10 nM induced persistent downregulation of all 3 genes. E2 induced upregulation across the dose range in XY HUVEC that returned to near baseline by 7 days (Fig. 3Cright).
Sex-specific effects of E2 and DHT on gene expression HRMVEC were similar but more persistent over the 7-day culture period. DHT induced greater upregulation of ATP1A1, HSPD1, and CYCS in XY HRMVEC compared to XY HUVEC. CYCS expression returned to near baseline levels by 7 days at 0.01 nM, 1 nM, and 10 nM DHT, and HSPD1 expression returned to near baseline at 0.01 nM and 0.1 nM DHT. E2 again induced slight upregulation of all 3 genes at 0.1 nM that returned to baseline by 7 days. E2 induced downregulation in XY HRMVEC at 1 and 10 nM (Fig. 3Dleft). 0.01 nM DHT induced a slight upregulation of all genes in XX HRMVEC at 48 hours, but this transitioned to a downregulation at 72 hours and 7 days. E2 induced upregulation of HSPD1, ATP1A1, and CYCS in all XX HMRVEC groups, with a return to near baseline levels only in the 0.1 nM E2 group at 7 days (Fig. 3Dright). For all cell types, significant fold changes in gene expression at early time points tended to decrease by 7 days (Figure S5, S6).
Sex-specific hormone effects on mitochondrial membrane potential
The tetramethylrhodamine (TMRM) dye assay was used to assess mitochondrial membrane potential. TMRM dye accumulates in mitochondria at a rate proportionate to the mitochondrial membrane potential58. Accordingly, the intensity of TMRM fluorescence signals can be indirectly extrapolated to mitochondrial function59. We hypothesized that sex-matched hormone stimulation would increase mitochondrial membrane potential in line with the trends seen in ATP1A1 and CYCS mRNA expression (Fig. 4). The TMRM signal intensity in XX HLF and XY HLF were roughly equivalent at baseline and the sex-specific response to E2 and DHT largely mirrored gene expression results (Fig. 4A). XY HLF showed a non-significant increase in TMRM signal intensity at 0.1 nM E2 that then decreased in a dose-dependent manner, resulting in a significant decrease at 10 nM E2. DHT significantly increased the TMRM signal in XY HLF at 0.1 nM DHT, 1 nM DHT, and 10 nM DHT (Fig. 4B). E2 significantly increased the TMRM signal intensity in XX HLF in a dose-dependent manner, while DHT significantly decreased signal intensity at all doses (Fig. 4C). HOF followed the same trends measured in HLF but to varying levels of significance (Fig. 4D). 0.1 nM E2 caused a non-significant increase in the TMRM signal intensity of XY HOF, while higher levels of E2 again decreased the TMRM signal, but this difference was not significant. DHT effects on XY HOF mirrored those measured in XY HLF, with 0.1 nM, 1 nM, and 10 nM DHT all significantly increasing the TMRM signal (Fig. 4E). In contrast to measured trends in XX HLF, E2 did not increase the TMRM signal of XX HOF at any of the tested doses. However, DHT significantly decreased the TMRM signal intensity of XX HOF in a dose-dependent manner (Fig. 4F).
A Representative micrographs of XY (top row) and XX (bottom row) human lung fibroblast (HLF) monolayers probed with TMRM fluorescent dye (red) after exposure to varying concentrations of E2 and DHT over 48 hours. Scale bar = 200 μm. B Mean fluorescent intensity of the TMRM signal normalized to XY HLF cell area. After 48 h of culture in E2 doses of 0, 0.1, 1, and 10 nM and DHT doses of 0.01, 0.1, 1, and 10 nM. The same dose responses are shown for all other plots of TMRM intensity. C Mean fluorescent intensity of the TMRM signal in XX HLF after 48 h of culture with the indicated doses of E2 and DHT. D Representative micrographs of XY (top row) and XX (bottom row) human ocular fibroblast (HOF) monolayers probed with TMRM dye (red) after exposure to varying concentrations of E2 and DHT for 48 hours. Scale bar = 200 μm. E Mean fluorescent intensity of the TMRM signal in XY HOF after 48 hours of culture with the indicated doses of E2 and DHT. F Mean fluorescent intensity of the TMRM signal in XX HOF after 48 h of culture with the indicated doses of E2 and DHT. n = 4 donors per sex for HLF, 3 donors per sex for HOF, 3 wells per condition, 3 images per well. B, C, E, F analyzed via two-way ANOVA between control and experimental groups. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Bars represent SEM. Dots represent per donor average.
In endothelial cells, TMRM assays similarly corroborated gene expression changes (Fig. 5A, D). E2 significantly decreased the TMRM signal of XY HUVEC at 1 nM, and all doses of DHT significantly increased the TMRM signal intensity (Fig. 5B). E2 induced a non-significant trend of increasing TMRM signal in XX HUVEC. DHT significantly decreased the TMRM signal of XX HUVEC at 0.1 nM and 10 nM (Fig. 5C). In line with results in the other cell types, E2 decreased the TMRM signal in XY HRMVEC at 1 nM and 10 nM, while only 1 nM DHT significantly increased the TMRM signal (Fig. 5E). E2 induced a significant increase in TMRM signal in XX HRMVEC at 10 nM, with a trend of insignificant increases at lower doses. In line with results in HUVEC, DHT significantly decreased the TMRM signal intensity in XX HRMVEC at 1 and 10 nM (Fig. 5F). Taken together, these data further support that E2 and DHT exert sex-specific effects in defined culture medium, as demonstrated by the significant modulation of gene expression and bioenergetic capacity in multiple types of primary human fibroblasts and endothelial cells of both sexes.
A Representative micrographs of XY (top row) and XX (bottom row) human umbilical vein endothelial cells (HUVEC) monolayers probed with TMRM fluorescent dye (red) after exposure to varying concentrations of E2 and DHT over 48 hours. Scale bar = 200 μm. B Mean fluorescent intensity of the TMRM signal normalized to XY HUVEC cell area. After 48 h of culture in E2 doses of 0, 0.1, 1, and 10 nM and DHT doses of 0.01, 0.1, 1, and 10 nM. The same dose responses are shown for all other plots of TMRM intensity. C Mean fluorescent intensity of the TMRM signal in XX HUVEC after 48 h of culture with the indicated doses of E2 and DHT. D Representative micrographs of XY (top row) and XX (bottom row) human retinal microvascular (HRMVEC) monolayers probed with TMRM dye (red) after exposure to varying concentrations of E2 and DHT for 48 h. Scale bar = 200 μm. E Mean fluorescent intensity of the TMRM signal in XY HRMVEC after 48 h of culture with the indicated doses of E2 and DHT. F Mean fluorescent intensity of the TMRM signal in XX HRMVEC after 48 h of culture with the indicated doses of E2 and DHT. n = 4 donors per sex for HUVEC, 3 donors per sex for HRMVEC, 3 wells per condition, 3 images per well. B, C, E, F analyzed via two-way ANOVA between control and experimental groups. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Bars represent SEM. Dots represent per donor average.
Sex-specific effects of E2 and DHT on bulk tissue vasculogenesis
A simple culture device that enables the generation of 3D tissue discs with a constrained volume was used to investigate sex-specific effects of E2 and DHT on bulk tissue vasculogenesis (Figure S7H)60. These devices were crafted from polydimethylsiloxane (PDMS), a common material used in organ chip applications with known hydrophobicity and propensity to absorb small molecules61,62. We employed a simple surface coating method via the incorporation of a PEG-PDMS self-sorting polymer to prevent hormone absorption63,64. A Nile Red absorption test was performed to assess the efficacy of PEG incorporation for preventing absorption of small hydrophobic molecules61. Nile Red has a theoretical octanol-water partition coefficient (Kow) of log Kow = 4.38 and E2 has a similar experimental Kow of log Kow = 4.01 (theoretical log Kow = 3.94). The similarity of partition coefficients and molar masses makes Nile Red an appropriate analog for modeling E2 absorption65,66,67. Nile Red absorption tests show that the presence of the PEG-copolymer significantly decreased absorption to only background levels of fluorescence, which suggests that these devices can be successfully used with minimal absorption of E2 or DHT from media formulations (Figure S7A–G). Sex-matched HUVEC and HLF embedded in a collagen type I and fibrin hydrogel blend seeded in the culture device undergo de novo vascular network formation that is quantified by morphometric analysis. Gels were cultured in typical culture media supplemented with aprotinin for 48 h and then switched to charcoal-stripped serum for a 48 h starvation period before exposure to a dose range of E2 and DHT for an additional 96 h. Control devices were treated with ethanol vehicle at the same concentration as hormone-treated tissues. Fixed and stained tissues were analyzed via the open-source vascular network morphometry tool REAVER37.
E2 qualitatively enhanced vasculogenesis in XX tissues compared to baseline controls as seen in representative micrographs (Fig. 6A). Total vessel length and branchpoint count were significantly increased as compared to vehicle baselines at 1 nM and 10 nM E2 (Fig. 6B, C). Mean segment diameter significantly increased and maximum diffusion distance significantly decreased at 0.1 nM E2, 1 nM E2, and 10 nM E2 (Fig. 6D, E). DHT did not induce any significant changes from baseline state except for decreased branchpoint counts at 10 nM DHT (Fig. 6B–E). DHT induced a qualitative increase in rounded endothelial cells that was quantified as a significant increase in non-participating endothelial cells (Fig. 8A). By contrast, there was no significant change in non-participating endothelial cells in E2-treated XX tissues. Collectively, these data suggest that E2 accelerates and enhances vascular network formation in XX tissues, while DHT may inhibit cell adhesion and spreading.
A Representative maximum intensity projections of XX vascular tissue cultured for 7 days total. Gels were allowed to form under normal conditions for 48 hours, hormone starved in charcoal stripped media for 24 hours, and then exposed to the indicated levels of E2 or DHT for 4 days. Whole mount staining with DAPI (blue), F-actin (green), and endothelial-specific lectin UEA-1 (red). B–E Morphometric analysis of vasculogenesis. B Total vessel length in micrometers (μm) provides a measure of the magnitude of vascular network formation. C Branchpoint count provides a measure of network complexity. D Mean segment diameter in micrometers (μm) provides a measure of average vessel caliber. E Maximum diffusion distance in micrometers (μm) represents half of the average distance between adjacent branches of the vascular network and provides a measure of network density and uniformity spacing. Line represents mean. Compared via two-way ANOVA. n = 3 donor combinations per sex, 3 gels per condition, 3 images per gel. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Dots represent donor pair average.
XY tissues exhibited relatively incomplete vasculogenesis with lower baseline metrics than those of XX models (Figs. 6, 7). E2 exposure inhibited XY tissue vasculogenesis, as quantified by significantly decreased total vessel length and mean segment diameter at 1 nM E2 and 10 nM E2 (Fig. 7B–E). DHT significantly increased branchpoint counts at 0.1 nM and 1 nM, and mean segment diameter at 0.1 nM (Fig. 7C, D). The only other significant hormone effects in XY tissues were increased non-participating cell counts. DHT and E2 both significantly increased non-participating cell counts in XY tissues (Fig. 8B, D). The vasculogenesis study collectively demonstrated that E2 and DHT exert sex-specific effects on tissue morphogenesis, with general trends of enhancement and inhibition that match results of 2D culture assays.
A Representative maximum intensity projections of XY vascular tissue cultured for 7 days total. Gels were allowed to form under normal conditions for 48 hours, hormone starved in charcoal stripped media for 24 hours, and then exposed to the indicated levels of E2 or DHT for 4 days. Whole mount staining with DAPI (blue), F-actin (green), and endothelial-specific lectin UEA-1 (red). B–E Morphometric analysis of vasculogenesis. B Total vessel length in micrometers (μm) provides a measure of the magnitude of vascular network formation. C Branchpoint count provides a measure of network complexity. D Mean segment diameter in micrometers (μm) provides a measure of average vessel caliber. E Maximum diffusion distance in micrometers (μm) represents half of the average distance between adjacent branches of the vascular network and provides a measure of network density and uniformity spacing. Box spans from minimum to maximum. Line represents mean. Compared via two-way ANOVA. n = 3 donor combinations per sex, 3 gels per condition, 3 images per gel. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 Dots represent donor pair average.
A, B DHT significantly increases the number of non-participating cells indicating disruption of vascular network formation in both XX (A) and XY (B) tissues at doses above 0.01 nM. C, D E2 does not significantly alter the number of non-participating endothelial cells participating in the vascular networks of XX (C) tissues, but significantly increases the number of non-participating cells in XY (D) tissues at all doses tested. Compared via two-way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n = 3 donor pairs per sex, 3 gels per condition, 3 images per gel. Dots represent donor pair average.
Discussion
Design of sex-specific cell culture methods and microphysiological systems
Primary human cell cultures increase the translational relevance of assay readouts by preserving some features of their respective in vivo phenotype68,69,70,71,72,73. Microphysiological systems (MPS) are bioengineered culture systems that situate primary human cells in a 3D culture environment to mimic the layered tissue architectures seen in vivo and grant control over biophysical parameters like mechanical forces and concentration gradients27,74,75,76. There are established methodologies for guiding biological processes like vasculogenesis and angiogenesis in MPS, but none of these methods account for biological sex77,78,79,80,81,82. More broadly, there are no standardized sex-specific protocols for 2D culture of primary human cells. The current study demonstrates that the interplay of chromosomal sex and sex hormones significantly modulates cellular bioenergetics, proliferation, gene expression, and tissue-scale processes in multiple types of cultured primary human cells of both sexes. The congruence of results obtained using fibroblasts (HLF, HOF, HDF) and endothelial cells (HUVEC, HMRVEC) from multiple sources suggests that sex-specific hormonal regulation of cellular physiology may be conserved in different organ systems, thereby enabling the development of generalizable sex-specific culture methods. However, hormone responses in other primary human cell types such as epithelial and neural cells must be defined before a framework of generalizable sex-specific culture methods can be constructed.
Biological sex contributes to the variability and heterogeneity of cells in culture14,71,83,84,85. While prior research has shown that hormones can influence cell growth and proliferation, most studies use cells of a single sex or immortalized cells with aberrant sex hormone signaling machinery17,42,86,87,88,89. Moreover, many culture methods rely on media formulations containing hormonally active fetal bovine serum (FBS) that may affect the readouts of 2D and 3D culture assays in a sex-specific manner90,91,92. Serum-free media formulations and charcoal-stripped FBS are alternatives to FBS-containing media93,94,95,96,97, but these methods lack other physiologically relevant components of FBS98,99,100. While there is limited precedent for our study, previous literature reported that androgen stimulation amplified inflammatory responses of female HUVEC, while DHT promoted male HUVEC proliferation in line with our results84,101,102. Other studies reported that E2 upregulates collagen production in HDF and other fibroblasts103,104,105,106. Taken together with this prior work, our study highlights that it is not adequate to simply consider and report the chromosomal sex of cells. The hormonal composition of culture medium must be carefully crafted to capture sex-specificity, as needed for translationally relevant modeling of biological sex differences in culture.
Sex-specific effects of E2 and DHT on bioenergetics and proliferation
Proper mitochondrial function and ATP generation fuel cell proliferation107,108,109,110,111,112. We have shown that supplementation with sex-matched hormone concentrations, i.e., E2 in XX cells and DHT in XY cells, significantly increases proliferation (Figs. 1, 2). Indeed, increased proliferation was correlated with evidence of sex hormone enhancement of bioenergetic capacity (Figs. 3–5). Preclinical animal models and clinical studies have shown that mitochondrial function and ATP production, both central to cellular bioenergetics, are tied to hormonal status. In male rodents, testosterone administration increases mitochondrial function and ATP levels in both brain113,114,115,116 and muscle117,118,119,120. Similar trends are documented in the effects of estrogens in female patients. E2 enhances mitochondrial respiration and increases glucose transporter expression within the female brain121,122,123,124,125, and ovariectomy causes E2-reversible metabolic dysfunction in female mice126,127. Considering the universal role of energy production in cellular physiology, tuning sex-matched hormone concentrations in culture medium is a tractable approach to enhance the performance of primary human cells in a variety of culture systems and bioassays.
Cross-sex hormone stimulation, or E2 in XY cells and DHT in XX cells, downregulated mitochondrial membrane potential, decreased proliferation, and decreased expression of genes associated with energetic capacity (Figs. 1–5). These early data from our culture systems are congruent with clinical observations. Androgen excess in females and estrogen excess in males are both known to cause adverse metabolic conditions and increased risk of cardiovascular diseases22,128,129,130,131,132,133,134,135. In female rats with polycystic ovarian syndrome, androgen excess results in downregulation of electron transport and decreased ATP production in ovarian tissues136,137. While our simplified single-hormone models do not recapitulate the complex endocrinology of cross-sex hormone therapy, this paradigm serves as a basis for further development of models to evaluate the roles of sex hormones in cardiovascular physiology and disease.
Influence of sex on the vascularization of engineered tissues
Stimulating vascularization is a critical aspect of engineering 3D tissues for potential therapeutic applications and in the development of physiologically relevant tissue surrogate models such as MPS79,138,139,140,141. We show that E2 enhances vasculogenesis in XX tissues (Figs. 6, 8), suggesting that E2 supplementation is a tractable approach to create more robust tissue-engineered female vascular networks. However, inhibition of vasculogenesis by E2 in XY tissues suggests that the pro-vasculogenic role of high E2 concentrations is sex-specific (Figs. 7, 8). Importantly, physiological levels of E2 do seem to be beneficial in males, as supported by clinical observations that dysregulated estrogen signaling via either estrogen receptor mutations or aromatase receptor mutations results in an increased risk of cardiovascular disease in male patients142,143,144,145,146. The non-significant response of male tissues to DHT exposure suggests that the role of androgens in vascular tissue engineering is more nuanced, potentially due to the absence of necessary concomitant estrogen signaling in male patients as mentioned above (Fig. 7). These observations are congruent with clinical trends showing that proper levels of matched hormones can be protective to cardiovascular health. It is well-documented that E2 is protective in female patients147,148,149,150, and low levels of testosterone are a known risk factor for cardiovascular disease in male patients151,152,153. Clinically, cross-sex hormone therapies have been correlated to an increased incidence of various cardiovascular pathologies. Female to male transgender patients on androgens exhibit increased arterial stiffness and elevated blood pressure154,155,156,157. Male to female patients on estrogens exhibit adverse lipid profiles, increased risk of metabolic disorders, and increased risk of cardiovascular disease155,156,158,159.
Perspectives and significance
Our findings suggest that in vitro culture systems can be engineered to evaluate sex-specific differences in cell biology and in MPS. While these experiments were designed to evaluate E2 and DHT concentrations spanning the physiological range of reported serum levels, E2 or DHT alone do not recapitulate the in vivo sex hormone profile22,160,161. While E2 is often thought of as a female hormone and DHT as male, both hormones are present and active in individuals of both sexes, albeit at differing levels with E2 being markedly higher in females and DHT being higher in males22. The levels of hormones used herein are in line with the average premenopausal female levels of estradiol and the average male levels of DHT; the lowest concentrations of each (i.e., 0.01 nM DHT and 0.1 nM E2) represent near physiological conditions for cross-sex hormones22. Future work will evaluate the effects of physiological ratios of E2 and DHT concurrently, concentrations of hormones that represent post-menopausal conditions, and additional estrogens and androgens beyond E2 and DHT that are known to be important modulators of physiology and disease25,162,163,164. Additionally, while androgens may impact cells directly via androgen receptor signaling, testosterone is also often converted to estrogens prior to cell signaling via aromatase165,166,167. Importantly, DHT is considered a pure androgen as it cannot be aromatized to estrogen168, so we can reasonably conclude that effects of DHT measured in the current study are due to androgen signaling. In conclusion, the results presented herein establish a strong rationale for developing sex-specific culture methods to facilitate in vitro modeling of sex differences in physiology and disease.
Data availability
Data is available at https://doi.org/10.6084/m9.figshare.28774421.v1. Any other data and more detailed explanations of laboratory procedures will be made available upon reasonable request to the corresponding author. All CAD design files are made available in the Supplementary Information for this article.
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Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM152305. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Prof. Sarah Lindsey of the Tulane Center of Excellence in Sex-based Precision Medicine for helpful discussions that contributed to the experimental designs and interpretation of the data reported in this study.
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A.T.M. helped conceive the study, led all aspects of the experimental work and data analysis, and participated in writing and revision of the manuscript. Y.V.M. contributed to 2D cell culture studies and associated data analysis. K.M.C. contributed to 3D vasculogenesis studies and morphometric analysis. D.A.C. contributed to 2D cell culture studies and associated data analysis. F.M.J. helped conceive the study and participated in writing and revision of the manuscript. M.J.M. conceived the study, directed all aspects of the research, and wrote the manuscript.
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Martier, A.T., Maurice, Y.V., Conrad, K.M. et al. Estradiol and dihydrotestosterone exert sex-specific effects on human fibroblast and endothelial proliferation, bioenergetics, and vasculogenesis. Commun Biol 8, 1422 (2025). https://doi.org/10.1038/s42003-025-08822-1
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DOI: https://doi.org/10.1038/s42003-025-08822-1
