Human HF organ culture and hair cycle scoring
Human scalp skin specimens were obtained from dermatology patients after obtaining informed consent, following the Declaration of Helsinki principles. The study was approved by the Institutional Review Board of the CHA Bundang Medical Center (IRB No. CHAMC 2018-09-009).
Anagen human HFs were isolated by microdissection and maintained in William’s E medium (WelGENE, South Korea) supplemented with 10 µg/ml insulin (Sigma-Aldrich, St. Louis, USA), 10 ng/ml hydrocortisone (Sigma-Aldrich), 20 mM HEPES (Invitrogen-Gibco-BRL, Grand Island, NY), and 1× antibiotic-antimycotic (Invitrogen-Gibco-BRL) for 1 day.
Isolated human HFs were exposed to 10-6 M CRF. On every second or third days, the medium was replaced, and human HFs were photodocumented. The hair cycle stages of cultured human HFs were scored every day according to the hair cycle scoring guidelines (25-27).
Cell culture and treatment
Three individual patients originated cells were used for the studies. Human DPC culture was established according to the method of Warren (44) with modifications. Cells were cultured in CnT Basal medium 2 containing two supplements (CnT-05.A and CnT-05.B) (CELLnTEC, Huissen, The Netherlands) with 1× antibiotic-antimycotic and incubated at 37 ℃ under 5% CO2.
For human ORSC culture, HFs were isolated from the dermis and attached to dishes coated with collagen type I. Outgrowing ORSCs were cultured in Keratinocyte SFM (Invitrogen-Gibco-BRL) at 37 °C under 5% CO2.
Cells were exposed to varying concentrations of CRF for up to 72 hours at 37 ℃. Materials including receptor antagonists were added one hour before hormone treatments. Antalarmin (Sigma-Aldrich) and astressin 2-B (Sigma-Aldrich) were used as CRFR1- and CRFR2-specific antagonists, respectively.
Immunocytochemistry
Cells were fixed with 4% formaldehyde for 10 min, washed with PBS and blocked with 5% BSA in PBS for one hour. The slides were incubated with each primary antibody in blocking solution. After the cells were washed, they were labeled with secondary antibody with fluorescent dye for one hour. Then, the cells were immediately mounted with DAPI solution and visualized with fluorescence microscopy (Thermo Fisher Scientific, USA). The antibodies used in immunocytochemistry are listed in Table 1.
Cell proliferation
The changes in cell number were assessed using an EZ-Cytox assay kit (Daeil Lab Service, Korea) according to the manufacturer’s instructions. Human DPCs were seeded at a density of 7,000 cells per well in 48-well plates. After attachment, the cells were treated with each test material for 3 days. Medium was replaced with supplement-free CnT basal medium with 20 µl/well of EZ-Cytox solution. After 2 hours, the absorbance was measured at 450 nm using a Synergy H1 Multi-Mode Reader (Biotek, USA). The optical density (OD) of each well was used to calculate the cell number based on the authentic standard curves.
Measurement of intracellular reactive oxygen species (ROS)
Cells were washed with DPBS (WelGENE) and stained with 2',7'-dichlorodihydrofluorescein diacetate acetyl ester (H2DCFDA, Thermo Fisher Scientific) for 30 min at 37 ℃ in dark conditions. Guava EasyCyte (Merck Millipore, Germany) was used for analysis.
Cell cycle analysis
Cells were washed with DPBS (WelGENE), fixed with 80% cold ethanol for 2 hours, centrifuged to remove the ethanol, and washed with cold DPBS 2 times. Cells were stained with PI/RNase in DPBS containing 1% FBS (HyClone, USA) for 30 min at 37 °C. Guava EasyCyte (Merck Millipore) was used to analyze the cell cycle.
Apoptotic cell analysis
CRF-exposed cells were analyzed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. Annexin V is used to identify apoptotic cells, and PI is used to distinguish viable from nonviable cells. Guava EasyCyte (Merck Millipore) was used for analysis.
Semiquantitative RT-PCR
Total RNA was extracted from human DPCs using an RNA/protein one step kit (MACHEREY-NAGEL, Dueren, Germany) according to the manufacturer’s protocol. cDNA was synthesized from 2 µg of total RNA using reverse transcriptase, oligo dT and dNTPs and was used as the template for PCR. The cDNA was amplified in a PCR reaction containing 10 pM of specific primers using a Veriti 96 Well Thermal Cycler (Applied Biosystems, USA). Amplification consisted of 28~35 cycles as follows: denaturation at 95 ℃ for 5 min, annealing at 56.2~62 ℃ for 30 sec and a final extension at 72 ℃ for 5 min. The PCR products were separated by 2% agarose gel electrophoresis and visualized with a Gel DocTM EX Imager (Bio-Rad, USA). All primers used are described in Table 2.
Western blot analysis
Total protein was extracted from human DPCs using an RNA/protein one step kit (MACHEREY-NAGEL) according to the manufacturer’s protocol. The proteins were separated by electrophoresis in 10% SDS-PAGE gels and transferred to 0.2 µm pore size PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in Tris buffered saline-Tween 20 (TBST 0.1% Tween 20) for an hour and then incubated with each primary antibody overnight at 4 °C. After three washes, the membranes were incubated with secondary antibody for one hour. After the samples were washed 5 times, immunoreactive protein was visualized with ClarityTM Western ECL Substrate (Bio-Rad) and detected using Amersham HyperfilmTM ECL (GE Healthcare, UK). Experiments were performed three times. The antibodies used in the western blot analyses are listed in Table 1.
cAMP assay
Human DPCs were incubated (5 × 105 cells/100 mm dish) for 30 min at 37 ℃ in media containing 0.5 mM of phosphodiesterase inhibitor and 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich). Then, CRF was added to media containing 0.5 mM of IBMX, and the cells were incubated for one hour at 37 ℃. The cAMP concentration was measured in cultured human DPCs using a Cyclic AMP ELISA Kit (Cayman, Michigan, USA) according to the manufacturer’s protocol. A Synergy H1 Multi-Mode Reader (Biotek) was used to measure the cAMP concentrations, and the concentration was calculated from the standard curve.
ACTH assay
Cells were seeded at a concentration of 3 × 105 cells per 100 mm dish. After attachment, the cells were treated with CRF receptor antagonists and then exposed to CRF. ACTH was measured with an ACTH ELISA kit (MD Bioproducts, Switzerland) according to the manufacturer’s protocol. The absorbance was measured with a Synergy H1 Multi-Mode Reader (Biotek). The OD of each well was used to calculate the ACTH concentration based on the authentic standard curves.
Cortisol assay
Cells were seeded at a concentration of 3 × 105 cells per 100 mm dish. Cells were pretreated with antalarmin and astressin 2-B for one hour. CRF was added to the media. Cells were collected and washed with cold PBS and then lysed with lysis buffer. Cortisol levels were measured in cultured human DPCs using a Parameter cortisol kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. The absorbance was measured using a Synergy H1 Multi-Mode Reader (Biotek). The OD of each well was used to calculate the cortisol concentration based on the values of the authentic standard curves.
Statistical analysis
Statistical analysis was performed using a two-tailed Student’s t-test to compare the experimental groups. P-values were considered to be significant at P<0.05 or P<0.01.