Figure 2

Co-culture with VECs enhanced corticosterone secretion from NR5A1-induced steroidogenic cells. (A) Co-culture procedure overview. ADSCs (5.0 × 10⁴ cells/well) were inoculated with adeno-lacZ (Ctrl) or adeno-NR5A1 (NR5A1) (MOI = 25). Three days after inoculation, 5.0 × 10⁴ NR5A1-ADSCs were co-cultured with 0, 5 × 10³, 2.5 × 10⁴, or 5 × 10⁴ of VECs, denoted as NR5A1, NR5A1:VEC = 1:0.1, NR5A1:VEC = 1:0.5, or NR5A1:VEC = 1:1, respectively. Subsequently, they were co-cultured for 4 days. (B) Corticosterone, and testosterone levels in the medium were measured using ELISA, and the ratio of corticosterone to testosterone (B/T) was calculated. (C), Ctrl-ADSCs, NR5A1-ADSCs, and NR5A1-ADSCs + VEC were cultured similarly, and cortisol and aldosterone were measured using LC–MS/MS. (D), Phase contrast images showing the morphology of NR5A1-ADSCs (upper panel) and NR5A1-ADSCs (lower panel) co-cultured with VECs. DsRed positive cells in each condition were counted and normalized by region of interest (n = 3, mean ± standard error). Statistical analyses were performed using Student’s t-test or one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ns; not significant). Scale bar = 100 μm. ADSCs, Adipose-derived stem cells; ANOVA, Analysis of variance; DHEA, Dehydroepiandrosterone; DsRed, Discosoma sp. red fluorescent protein; ELISA, enzyme-linked immunosorbent assay; LC–MS/MS, liquid chromatography-tandem mass spectrometry; MOI, multiplicity of infection; NR5A1, Nuclear receptor subfamily 5 group A member 1; VEC, vascular endothelial cells.