DPSCs culture and CM preparation

DPSCs (Lonza, Walkersville, USA) were characterized by surface marker profiling (negative: CD34, CD45, and CD133; positive: CD105, CD166, CD29, CD90, and CD73) performed by the manufacturer. DPSCs at passage 6–8 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, Saint Louis, USA) supplemented with 10% fetal bovine serum (Bovogen, Melbourne, Australia) and 1% penicillin–streptomycin solution (Fujifilm Wako, Osaka, Japan) at 37 °C in 5% CO2 until 80% confluency. Subsequently, these cells were washed with phosphate-buffered saline (PBS; Fujifilm Wako, Osaka, Japan), and the culture medium was replaced with the vehicle medium (serum-free DMEM). After 48 h of incubation in 21% and 1% O2 (normoxic [N-] and hypoxic [H-] conditions, respectively), the cells were collected and centrifuged at 400 × g and 4 °C for 3 min. The supernatants were collected and centrifuged at 1700 × g and 4 °C for 3 min. Following this, the supernatants resulting from this centrifugation were collected and defined as N-CM or H-CM. CM was stored at − 80 °C before being used in the subsequent experiments.

In vitro model: role of DPSC-CM in CYP-induced keratinocyte cytotoxicity

The NHEKs (Kurabo, Osaka, Japan) were cultured in a keratinocyte serum-free medium (KSFM; HuMedia-KG2, Kurabo, Osaka, Japan) according to the manufacturer’s instructions. They were used at passage 1–3 to ensure maximal proliferative capacity for all assays.

The NHEKs were seeded in 96-well culture plates and pre-treated with a mixture of 75 uL KSFM and 75 uL control-group medium (DMEM) or test-group media, i.e., vehicle (DMEM), N-CM, or H-CM, for 24 h in each well. These cells, except those in the control group, were subsequently stimulated by CYP (Endoxan, Shionogi, Osaka, Japan) for 48 h before being assayed; 50 µL of CYP dissolved in KSFM was added directly to the previous culture medium in each well. To avoid cell loss, NHEKs were centrifuged at 1500 RPM for 3 min, in the culture plates, before medium change or addition.

Cell viability assay

Cell viability was tested using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The 96-well culture plates were incubated for 2–3 h after adding Cell Counting Kit-8. The absorbance at 450 nm was subsequently measured using a microplate reader (Infinite M200pro, Tecan, Männedorf, Switzerland). Representative photomicrographs were taken (EVOS FL, Thermo-Fisher, Waltham, USA).

Lactate dehydrogenase cytotoxicity assay

The activity level of lactate dehydrogenase (LDH) released through the damaged plasma membrane of the NHEKs into the supernatant was assayed using the Cytotoxicity LDH Assay Kit-WST (Dojindo, Kumamoto, Japan). The culture supernatant was used to measure LDH, and the cells were used in the cell viability assay. The absorbance of the supernatant or the cells was subsequently measured using a microplate reader (Infinite M200pro, Tecan, Männedorf, Switzerland). The LDH activity level was defined as the ratio of the supernatant absorbance to the living cell absorbance. Each independent experiment comprised six technical replicates.

Determination of apoptosis

An In Situ Cell Death Detection Kit, TMR red (Roche, Basel, Switzerland) was used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, while DAPI (R37606, Life Technologies, Carlsbad, USA) was used for nuclear staining. The images were taken using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan).

In vitro: Cells were counted automatically using a BZ-X800 Analyzer (Keyence, Osaka, Japan). In each independent experiment, 20 different fields in one well of a 96-well plate were analyzed for one group.

In vivo: TUNEL staining was performed on 4-μm thick formalin-fixed paraffin-embedded sections. The skin tissue was divided equally into three sections (head, middle, and tail) for analysis. A minimum of 95 HFs from the dorsal skin at 100–200 × magnification was analyzed for each mouse.

In vivo CIA model

Six-week-old syngeneic female C57BL/6J mice were purchased from Charles River Japan (Tokyo, Japan). The mice were housed in standard cages on a 12 h light/dark cycle and were provided water and mouse chow ad libitum.

To minimize fluctuations in the HF cycle, dorsal skin HFs in the telogen phase were induced to enter the anagen phase immediately via depilation with a wax sheet (Veet, Reckitt Benckiser, Tokyo, Japan), confirmed by a pink skin color after a 7-day acclimatization period [31]. A single dose of 120 mg/kg CYP dissolved in 0.9% NaCl solution was injected intraperitoneally on day 9 post-depilation (p.d.) [29]. The mice were administered inhalation anesthesia (apparatus: Narcobit-e II, Natsume Seisakusho, Tokyo, Japan) before all procedures.

In the mouse model aimed at studying CIA progression and the damage-response pathway, a total of 54 mice, 18 in each group, were used. The vehicle (DMEM), N-CM, or H-CM were injected subcutaneously once a day from day 2 p.d. to days 14, 16, or 32 p.d., respectively, when the mice were euthanized for various purposes. The mice were euthanized with a carbon dioxide inhalation device and the fill rate of carbon dioxide was standardized by the experimental animal division of Nagoya University. If both cessation of breathing and ocular pallor were observed after initiation of carbon dioxide inhalation, the mice would continue to be exposed to carbon dioxide for an additional passive exposure time of 3 min, which reliably resulted in irreversible euthanasia for mice.

In the mouse model aimed at studying post-CIA hair regrowth, a total of 18 mice, 6 in each group, were used. The vehicle (DMEM), N-CM, or H-CM were injected subcutaneously daily from day 15 p.d. to day 59 p.d. when the mice were euthanized by carbon dioxide inhalation. The microstructure of the hair surface was observed using a scanning electron microscope (SEM).

Macroscopic analysis

Images of mouse dorsal skin were first converted to 8-bit format and subsequently analyzed for CIA and hair regrowth using ImageJ (National Institutes of Health, Bethesda, USA). The grayscale (0–255) threshold was uniformly set, with 0–35 and 36–255 deemed as dorsal skin with black hair and with alopecia or gray hair, respectively. Before day 16 p.d., the mice had almost no gray hair, and all hair shafts were black. Here, areas of grayscale 0–35 and 36–255 were considered as dorsal skin with hair and alopecic skin, respectively, before day 16 p.d..

Quantitative histological analysis

Histological analysis of HFs was performed in sections stained with hematoxylin and eosin. Fresh skin tissue was washed with PBS and fixed with 10% formalin neutral buffer solution for 24 h. Tissue specimens were automatically processed for 21 h by a Tissue-Tek VIP 6 tissue processor (Sakura, Tokyo, Japan) and subsequently embedded in paraffin. The samples were cut equally into three sections (head, middle, and tail) and sliced into 4-μm slices before deparaffinization, hydration, and staining. A minimum of 78 dorsal skin HFs at 100–400 × magnification was analyzed for each mouse. The HF was identified and classified according to its hair cycle stage and signs of follicle dystrophy [28].

Determination of mRNA expression

Fresh skin samples were washed with PBS, transferred directly into a pre-chilled (2–8 °C) RNAlater Stabilization Solution (Invitrogen, Waltham, USA), and stored at 4 °C for 48 h. The samples were homogenized automatically using a Multi-Beads Shocker (Yasui, Osaka, Japan) in the presence of liquid nitrogen. For reverse transcription-quantitative PCR (RT-qPCR), the total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, USA). Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan), while RT-qPCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) driven by the AriaMx Real-Time PCR System (Agilent, Santa Clara, USA). Gapdh was used as the reference gene. The primers, designed by Primer Premier 5 (Premier Biosoft, San Francisco, USA), are listed in Additional file 1: Table S1.

Caspase-3/7 activity assay

Fresh skin samples were washed with PBS, transferred directly into a pre-chilled (2–8 °C) MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany), and stored at 4 °C for 24 h. The samples were homogenized automatically using the Multi-Beads Shocker (Yasui, Osaka, Japan) in the presence of liquid nitrogen and subsequently using an ultrasonic homogenizer (As One, Osaka, Japan), with a 20 × cell lysis buffer (200 mM TRIS, pH 7.5, 2 M NaCl, 20 mM EDTA, and 0.2% TRITON X-100). Thereafter, the samples were diluted 20 × to 1 ×. Next, the protein concentration of the samples was adjusted to a uniform concentration using the BCA Protein Assay Kit (Pierce, Thermo Scientific, Waltham, USA). Caspase-3/7 activity was assayed using the EnzChek Caspase-3 Assay Kit #1 (Molecular Probes, Eugene, USA). The fluorescence (excitation/emission 342/441 nm) was measured by a fluorescence microplate reader (Cytation 5, Biotek, Winooski, USA).

Microstructure observation of the hair surface

The dried hair specimens were fixed onto a brass stub before being coated with an osmium plasma coater (NL-OPC80NS, Nippon Laser &Electronics, Nagoya, Japan). Microscopic observation was performed at 1000 × magnification with a SEM (JSM-7610F, JEOL, Tokyo, Japan). The dorsum of each mouse was divided equally into six regions, and a representative hair shaft from each region was selected for analysis.

Subcutaneous tumor model

Seven-week-old syngeneic female C3H/HeNCrl mice were procured from Charles River Japan (Tokyo, Japan). After a 1-week acclimatization period, 1.08 × 106 poorly immunogenic murine oral squamous cell carcinoma (SCC-VII) cells in 100 uL PBS were subcutaneously inoculated into the dorsal region of the mice. The initiation of the experiment was marked as day 0, corresponding to the point at which the tumor volume (1/2 × long diameter × short diameter2) reached 150 mm3. On days 0, 3, and 6, the mice were administered 300 uL of the vehicle (DMEM), N-CM, or H-CM via tail vein injections. Tumor volume was measured every 3 days. The mice were euthanized by carbon dioxide inhalation on day 21, and the tumors were resected and weighed.

Statistical analysis

Data were presented as mean ± SEM of at least three independent experiments. Statistical analysis was performed using Graphpad Prism, version 8.4.0 (Graphpad Software, Boston, USA). Group differences were analyzed by the Mann–Whitney U-test. A value of P < 0.05 was considered statistically significant.