Transcriptome Analysis of Dihydrotestosterone-treated 2D- and 3D-cultured Dermal Papilla Cells

Hair follicle growth inhibitory effect of dihydrotestosterone (DHT)-treated two-dimensional (2D)- and 3D-cultured DPCs were evaluated. 2D- and 3D-cultured DPC proliferation was inhibited after co-culturing with outer root sheath (ORS) cells under DHT treatment. Moreover, gene expression levels of ß-catenin and neural cell adhesion molecule (NCAM) were significantly decreased and those of cleaved caspase-3 significantly increased in 2D- and 3D-cultured DPCs with increasing DHT concentrations. ORS cell proliferation also significantly increased after co-culturing in the control-3D model compared with the control-2D model. ki67 downregulation and cleaved caspase-3 upregulation in DHT-2D and 3D groups significantly inhibited ORS cell proliferation. Sequencing showed an increase in the expression of genes related to extracellular matrix synthesis in the 3D model group. Additionally, the top 10 hub genes were identified, and the expression of nine chemokine-related genes in DHT-treated DPCs was found significantly increased. We also identified the interactions between transcription factor (TF)–genes and microRNAs with hub genes and the TF–microRNA coregulatory network. Overall, the findings indicate that 3D-cultured DPCs are more representative of in vivo conditions than are 2D-cultured DPCs, and contribute to our understanding of the molecular mechanisms underlying androgen-induced alopecia. Overall design: Human hair follicles were taken from the balding (frontal) area of male patients undergoing hair transplantation surgery. Ethical approval and informed consent were obtained preoperatively from Nanfang Hospital of Southern Medical University. Dermal papillae were isolated from the bulbs of hair follicles, plated in a cell culture flask (Corning Inc., Corning, NY), and cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Waltham, MA) supplemented with 1% (v/v) penicillin–streptomycin and 20% (v/v) fetal bovine serum (FBS; Gibco) at 37 °C and 5% CO2. Once the outgrowth reached 80% confluence, human DPCs were harvested by incubation with 0.25% (w/v) trypsin/EDTA (Gibco) and transferred to new culture dishes at a split ratio of 1:2. For 3D culture, DPCs were seeded in an ultra-low attachment 96-well plate (Corning Inc.) with DMEM supplemented with 1% (v/v) penicillin–streptomycin and 10% (v/v) FBS. Spheroids were formed for 1 d after seeding and were used in the following assays.