Animals
SPF 5-week-old C57BL /6 mice, SPF 5-week-old male BALB/C mice and SPF C57BL /6 mice(1-3d) were purchased from the Experimental Animal Centre at Southern Medical University (Guangzhou, China).
Isolation and Culture of pelage DP cells
After removing the hair shaft on the back with shaver and depilatory paste, carefully cleaned the mouse with 75% ethanol, cut off the full layer of dorsal skin and removed panniculus carnosus with toothed forceps. Then the dorsal skin was rinsed with PBS containing 1% Antibiotic-Antimycotic (Gibco) 3 times and incubated with 0.2% collagenase I (Sigma-Aldrich) in TESCA buffer (Solarbio) for 1 h at 37◦C. Rinsed with PBS containing 1% Antibiotic-Antimycotic 3 times, the pelage DP were scraped off with micro-scissors and blown evenly in PBS. Subsequently, centrifuging for 5 min at 1 000r/min, resuspending the pellet with 2ml 5% Ficoll (Leagene) solution. Cautiously add, dropwise, 2ml 5% Ficoll (Leagene) solution with pellet to a 15ml centrifuge tube containing 2ml 10% Ficoll (Leagene) solution. After density gradient sedimentation for 3 min at 200 r/min, collecting the lower part and centrifuged for 5 min at 1 000r/min. Then, resuspending the pellet with DMEM(Gibco) and adding slowly to a 15ml centrifuge tube containing 2ml 10% Ficoll (Leagene) solution. After density gradient sedimentation for 3 min at 150 r/min, collect the upper part and centrifuged for 5 min at 1 000r/min. Finally, resuspending the pellet with 20% culture medium and cultured in the T25 flasks (Corning) at 37℃ 5%CO2.(Figure S1). All the pelage DPCs were cultured to passage 2(P2) for the further experiments.
Isolation and culture of whisker DP cells
After cleaning with 75% ethanol, the whisker pads were cut off bilaterally with the scissor. whisker pads were rinsed with PBS, and then turned upside down and separated the follicle from the surrounding tissue using sharp forceps to expose the dermal side and hair follicle end bulbs. Then using a pair of blunt forceps to gently grip the most distal part of the hair follicle, before cutting away the end-bulb with micro-scissors and immediately placing in a sterile petri dish containing PBS. The dissected hair bulbs were rinsed with PBS and incubated with 0.2% collagenase I (Sigma-Aldrich) in TESCA buffer(Solarbio) for 1 h at 37◦C. After that, add a sufficient amount of culture medium, centrifuged (1 000 r/min, 5 min, room temperature), discard supernatant and re-suspended with 20% culture medium, cultured in 6cm culture dish. All the whisker DPCs were cultured to passage 2(P2) for the further experiments.
Isolation of neonatal mouse epidermal cells
Postnatal day 1–3 (P1-3) mouse skin was cut off, cleaned by 75% ethanol. Then, rinsed with PBS containing 1% Antibiotic-Antimycotic for 3 times, the skin was incubated with 0.1% dispase at 4°C for 24 hours. The epidermis was then peeled from the dermis, cut the epidermis into pieces and incubated in 0.25% trypsin (Gibco) for 10 min at 37°C to yield a single-cell suspension. Then, the single cell was resuspended with PBS after centrifuging at 1000r/min for 5min prepared for hair follicle reconstruction.
Observation on the morphology, adhesion and cell migration of dermal papilla
After observing the morphology of dermal papilla obtained from whisker and dorsal skin, DPCs were cultured for 7 days. The adhesion, migration, morphological characteristics and growth pattern of dermal papilla cells were observed.
Immunofluorescence of specific markers
DP specific markers expression.
DPCs、pelage DP cells and single cell were fixed with 4% paraformaldehyde for 20 minutes, washed three times with TBST (Solarbio), and permeabilized with 0.1% Triton X-100 for 7 minutes at room temperature. After blocking with 5% BSA for 1h, cells were incubated with a 1:200 dilution of Versican, Nestin, NCAM, ALP, Sox2 antibody (Bioss) overnight at 4°C, and subsequently incubated with a 1:200 dilution of 488-conjugated anti-rabbit IgG antibody (Abcam) for 1 hours. Coverslips were mounted on glass slides using ProLong® Diamond Antifade Mountant with DAPI (Life Technologies, USA) prior to imaging with a confocal laser scanning microscope.
Sox2 expression of primary DP spheres.
Whisker and pelage DP spheres were isolated according above methods. After isolation ,DP Spheres were fixed with 4% paraformaldehyde for 20 minutes, washed and centrifugated with TBST three times, then permeabilized with 0.1% Triton X-100 for 30 minutes at room temperature, washed and centrifugated with TBST three times. After blocking with 5% BSA for 1h, 1:200 dilution of Sox2 antibody (Bioss) overnight at 4°C and subsequently incubated with a 1:200 dilution of 488/594-conjugated anti-rabbit IgG antibody (Abcam) for 2 hours, washed and centrifugated with TBST three times. DAPI was incubated for 30 mins, washed and centrifugated with TBST three times, finally prepared to image with a confocal laser scanning microscope.
Three-lineage differentiation of pelage and whisker DPCs
Adipogenic differentiation and Oil Red O staining
Briefly, DPCs were seed in 6-well plate at a density of 5x10^5/well, cultured in DMEM with 10% FBS for 24h, changed the culture medium to mouse mesenchymal stem cell adipogenic differentiation medium 1 (BG sciences, BGM-2533) (ADP1:Basal medium, FBS, Glutamin, Pennicillin-Streptomycin, Insulin, IBMX, Rosiglitazone, Dexamethasone) for 72h, changed the medium to ADP2 (Basal medium, FBS, Glutamine, Pennicillin-Streptomycin, Insulin) and cultured for 24h. The two media were used alternately for 3–5 times, until enough lipid droplets appeared in the cells. Oil red staining were performed after differentiation. Cells were washed once with PBS, 4% paraformaldehyde fixed the cells for 30 mins, washed twice with PBS, incubated with Oil Red O staining solution at room temperature for 30 mins. Washed twice with PBS, then the cells were imaged using inverted microscope.
Osteogenic differentiation and alizarin red staining
Briefly, DPCs were seed in 6-well plate at a density of 5x10^5/well, cultured in DMEM with 10% FBS for 24h, changed the culture medium to mouse mesenchymal stem cell osteogenic differentiation medium (BG sciences, BGM-2522, include: basal medium, FBS, Glutamine, Pennicillin-Streptomycin, β-Glycerophosphate, Ascorbate Acid, Dexamethasone). The medium was changed every 3 days. After 4 weeks induction, Alizarin red staining were performed. Cells were washed once with PBS, 4% paraformaldehyde fixed the cells for 30 mins, washed twice with PBS, incubated with Alizarin red staining solution at room temperature for 30 mins. Washed twice with PBS, then the cells were imaged using inverted microscope.
Chondrogenic differentiation and Alcian blue staining
Briefly, DPCs were seeded in 6-well plate at a density of 5x10^5/well, When the fusion degree of stem cells reached 90%, performed trypsin digestion, centrifugation, supernatant removal, added mouse mesenchymal stem cell chondrogenic differentiation medium (BG sciences, BGM-2544, including: basal medium, Sodium pyruvate, ITS supplement, TGF-β3, Ascorbic acid, Proline, Dexamethasone, Pennicillin-Streptomycin), took half of the cell suspension (2x10^5 cells) ,centrifuged, loosened the centrifuge tube cap, cultured in incubator. Culture medium changed every 3 days. After 4 weeks induction,Alcian blue staining were performed. Cell clusters were fixed with neutral formaldehyde, routinely paraffin-embedded sections, dewaxing and rinsing, alcian blue staining solution dyed for 30 mins, stop staining, imaged using inverted microscope.
Hair follicle reconstitution assays
SPF 5-week-old male BALB/C mice were randomly divided into 3 groups (neonatal epidermal cells + pelage DP cells; neonatal epidermal cells + DPC group; neonatal epidermal cells alone group). The cells were re-suspended in 50 µL PBS. After successfully anesthetized each point were injected 100 µ L cell suspension(2.5x10^5 epidermal cells + 5x10^5 DPCs). Four weeks after injection, grafts were observed and taken for Paraffin sections.
Isolation and identification of pelage HF-MSC exosomes
The culture medium supernatant of pelage HF-MSC were collected, 300g centrifugation for 10mins, 2000g centrifugation for 10mins to remove cells. Took the supernatant and pass through a 0.22 µM filter to remove cell fragments and vesicles with a diameter greater than 220 nm. Transfer the filtered supernatant to a new centrifuge tube at 4 ℃ to 100000g ultracentrifugation for 70mins. After centrifugation, remove the supernatant, used precooled 1 × PBS resuspended at 4 ℃ and 100000g. ultracentrifugation for 70mins. After centrifugation, exosomes were obtained. Using nanoparticle tracking analysis (NTA) measured the particle size of pelage HF-MSC exosomes, Nanosight NS300 software version 2.3 (Malven PANalytical) were used to analysis. The exosome were observed by Transmission Electron Microscope (TEM). Specific markers CD9 and CD81 were verified by western blotting.
Injection of fluorescent labeled cells/exosomes and sections
The pelage HF-MSC were labeled with DIO (Invitrogen) and injected into the dorsal skin of mice. After 7 days, the back skin of mice was taken and frozen section were performed and imaged with confocal laser scanning microscope. Exosomes of pelage HF-MSC were labeled by DIL(Invitrogen) and co-incubated with mice whisker hair follicles. After 72h, the hair follicles were sampled and frozen section were performed, imaged with confocal laser scanning microscope.