Stem Cell

Establishment and characterization of matched immortalized human frontal and occipital scalp dermal papilla cell lines from androgenetic alopecia – Scientific Reports

Time Stamp: December 4, 2023 7:00 PM
Source Node: 368747

Isolation and culture of human hair follicle DP and ORS cells

Units of human hair follicles were obtained during hair transplantation from matching balding (frontal) and non-balding (occipital) scalps of patients by punch biopsy (1 mm). The Medical Ethical Committee of the Kyungpook National University Hospital approved this study (IRB Number KNUH 2021-09-006), and written consent was obtained from patients according to the Declaration of Helsinki Principles. DP and outer root sheath (ORS) keratinocytes were isolated as previously described¹¹. Briefly DP were isolated from the bulbs of dissected hair follicles, transferred onto plastic dishes coated with bovine type 1 collagen, and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 20% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2. The explants were left for 7 days, and the medium was changed every 3 days. After primary DP cell outgrowth had become sub-confluent, cells were harvested with 0.25% trypsin/10 mM EDTA in Hank’s balanced salt solution (HBSS) and were maintained in DMEM supplemented with 10% FBS. In the subsequent experiments, primary dermal papilla (DP) cells at passage 2 were used. The hair shaft and hair bulb region of the hair follicles (HFs) were removed to prevent contamination with other cells.

HF remaining after cutting the bulb were then placed in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS) in tissue culture dishes coated with Biocoat collagen type I (CORNING, Kennebunk, ME04043, USA). After 3 days of culture, the medium was replaced with keratinocyte growth medium, EpiLife (Gibco BRL, Gaithersburg, MD, USA), containing 1% antibiotic–antimycotic solution and 1% EpiLife defined growth supplement. Once the cells reached subconfluency, they were harvested using 0.25% trypsin/10 mM EDTA in phosphate-buffered saline (PBS) and maintained in EpiLife medium. Primary ORS cells from the second passage were used for the experiments in this study.

Construction of immortalized F and O DP cells

F and O DP cells at passage 1 were transfected with a pSV3neo plasmid (ATCC; Manassas, VA, USA) carrying SV40T-Ag and neomycin resistance and a pGRN145 plasmid (ATCC) carrying hTERT and hygromycin resistance. Briefly, cells (5 × 10⁵) were transfected with 1 μg pSV3neo or pGRN145 as an internal control using Microporator (Invitrogen, Carlsbad, CA, USA; pulse voltage 1100, pulse width 50). After 48 h, immortalized cells were selected with 5 μg/ml hygromycin B (Thermo Fisher) and 5 μg/ml G-418 (Sigma). SV40T-Ag and hTERT expression was evaluated by immunocytochemistry and RT-PCR analysis, respectively. Immortalized frontal and occipital DP cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) with 10% heat-inactivated fetal bovine serum and were used for 35–50 passages in this study.

Cell proliferation assay

Immortalized F and O DP cells were plated in 96-well plates (1,000 cells per well). Cells were measured 24, 48, and 72 h after seeding with a cell counting kit-8 (Donjindo, Kumamoto, Japan). The absorbance was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm. For direct counting methods, 10⁵ immortalized F and O DP cells were plated in 10 mm. After 24, 48, and 72 h, cells were harvested with 0.25% trypsin/10 mM EDTA in Hank’s balanced salt solution and counted. An EdU proliferation kit (Abcam) was used following the manufacturer’s protocol to evaluate DNA synthesis in live cells. Cells were cultured in a 96-well plate for 24 h, treated with 20 μM EdU, and cultured for 3 h. After activation using EdU Additive Solution Reaction Buffer, DNA proliferation was observed under a microscope.

β-galactosidase staining

A β-galactosidase staining kit (Cell Signaling) was used to evaluate cell senescence. Briefly, after fixing at room temperature for 10 min, washing twice with PBS, and incubating overnight in β-galactosidase staining solution at 37 °C, senescence cells were observed in blue color under a microscope.

RT-PCR

Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from 3 μg total RNA using ImProm-II™ reverse transcriptase kit (Promega, Madison, WI, USA). PCR was performed using a Taq polymerase and forward and reverse primers, and the product was confirmed under UV light after electrophoresis in 1% agarose. Real-time PCR was performed using Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with 50 ng cDNA, 10 pM primers, and Power SYBRR Green premix (Applied Biosystems). The cycling conditions for amplification were: 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s. Primer sequences are listed in Supplementary Table 1.

Immunocytochemistry and immunofluorescence staining

For the immunocytochemistry of SV40T-Ag, immortalized F and O DP cells were seeded on 8-well slides (Nunc Lab-Tek, Roskilde, Denmark) at passage 4 after a 24-h treatment with hygromycin B and G418. Cells were fixed in 4% paraformaldehyde containing 0.1% Triton X-100 for 10 min at room temperature. After 3% H₂O₂ treatment for 30 min, blocking was performed in 5% normal donkey serum (Abcam, Cambridge, UK) for 1 h, and samples were incubated with antibodies against SV40T-Ag (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. Cells were washed 3 times with PBS and incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody for 1 h. After washing with PBS, color developed using AEC, and counterstaining was performed with hematoxylin.

For the immunofluorescence staining, cells were seeded, fixed, and blocked, as described above. Next, cells were incubated with antibodies against a-SMA (R&D Systems, Minneapolis, MN, USA), cytokeratin 1–3 (KRT 8; Chemicon, Temecula, CA, USA), versican (Seikaguka Corporation, Tokyo, Japan), biglycan (R&D Systems), perlecan (Zymed Laboratories, San Francisco, CA, USA), and AR (Abcam, Cambridge, UK) at 4 °C overnight. After washing three times with PBS, cells were incubated for 1 h at room temperature with Alexa Fluor 488-labeled donkey anti-rabbit or mouse secondary antibody (Molecular Probes, Eugene, OR, USA). Subsequently, slides were washed with PBS and counterstained with 4,6- diamidino-2-phenylindole (DAPI) for 10 min.

CM of immortalized F and O DP cells and proteome profiler array

After seeding 10⁶ cells in a 10-mm plate, the medium was replaced with serum-free DMEM. After 24 h, the CM was harvested from F and O DP cells. The CM was concentrated using Amicon Ultra (Millipore) by centrifugation and filtration and applied to human hair follicles. After 6 days of CM treatment, the length of human hair follicles was measured. A proteome profiler array (R&D Systems, Minneapolis, MN, USA) was used to analyze growth factors displaying differences between immortalized Fal and O cells.

TUNEL and Ki-67 staining

A TUNEL kit (EMD Millipore, Billerica, MA, USA) was used following the manufacturer’s protocol to evaluate apoptotic cells. In brief, cells were fixed in 1% paraformaldehyde for 10 min and post-fixed in ethanol-acetic acid (− 20 °C) for 5 min. Slides were washed with PBS, incubated with working strength TdT enzyme at 37 °C for 1 h, and incubated with digoxigenin fluorescein-conjugated antibody for 30 min. Next, cells were incubated with antibodies against Ki-67 (Millipore) at 4 °C overnight. After washing with PBS, Alexa Fluor 555-labeled donkey anti-mouse secondary antibody was incubated for 1 h at room temperature. The slides were counterstained with DAPI.

3D culture of immortalized F and O DP cells

The 3D cultured cells were harvested and re-seeded in a 96-well Hydro Cell plate (Nunc, Rochester, NY, USA). After 24, 48, and 72 h, pictures were taken under a microscope, and RNA was isolated.

Statistical analysis

Data are expressed as means ± standard deviation (SD). ANOVA was used for statistical analysis of the data. P < 0.05 was considered statistically significant.