Sex-specific actions of estradiol and testosterone on human fibroblast and endothelial cell proliferation, bioenergetics, and vasculogenesis

Progress toward the development of sex-specific tissue engineered systems has been hampered by the lack of research efforts to define the effects of sex-specific hormone concentrations on relevant human cell types. Here, we investigated the effects of defined concentrations of estradiol (E2) and dihydrotestosterone (DHT) on primary human dermal and lung fibroblasts (HDF and HLF), and human umbilical vein endothelial cells (HUVEC) from female (XX) and male (XY) donors in both 2D expansion cultures and 3D stromal vascular tissues. Sex-matched E2 and DHT stimulation in 2D expansion cultures significantly increased the proliferation index, mitochondrial membrane potential, and the expression of genes associated with bioenergetics (Na+/K+ ATPase, somatic cytochrome C) and beneficial stress responses (chaperonin) in all cell types tested. Notably, cross sex hormone stimulation, i.e., DHT treatment of XX cells in the absence of E2 and E2 stimulation of XY cells in the absence of DHT, decreased bioenergetic capacity and inhibited cell proliferation. We used a microengineered 3D vasculogenesis assay to assess hormone effects on tissue scale morphogenesis. E2 increased metrics of vascular network complexity compared to vehicle in XX tissues. Conversely, and in line with results from 2D expansion cultures, E2 potently inhibited vasculogenesis compared to vehicle in XY tissues. DHT did not significantly alter vasculogenesis in XX or XY tissues but increased the number of non-participating endothelial cells in both sexes. This study establishes a scientific rationale and adaptable methods for using sex hormone stimulation to develop sex-specific culture systems.