Adipose-derived stem cells (ASCs) have stimulatory effects on dermal papilla (DP) cells to promote hair-growth [1
]. For example, ASCs secrete multiple growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factors (bFGF), which can increase the proliferation of DP cells [1
]. Festa et al. showed that adipocyte lineage cells drive hair cycling by contributing to the skin stem cell niche, and suggested that platelet-derived growth factor-A (PDGF-A) expression by immature adipocytes regulates follicular stem cell activity [2
]. Grafting of ASC-enriched adipose tissue (i.e., by injection of the stromal vascular fraction of lipoaspirate) has shown promise as an alternative approach to treating baldness in men and women [7
]. We have identified stimulators that enhance the hair-regenerative potential of ASCs in vivo. For instance, vitamin C and low-dose ultraviolet B (UVB) increased secretion of hair growth-promoting factors by ASCs and induced anagen in animal models [3
]. Of the potential stimulators tested, we found that platelet-derived growth factor-D (PDGF-D) exhibited the strongest effects on ASCs and increased secretion of growth factors via mitogen-activated protein kinase (MAPK) pathways [5
]. In addition, CAP-18
for cathelicidin antimicrobial peptide (LL-37) increased the secretion of growth factors and the hair-regenerative efficacy of ASCs via early growth response 1 (ERG1) protein and the MAPK pathway [8
Minoxidil was first developed to treat male- and female-pattern alopecia. In addition to vasodilation, there is strong evidence that minoxidil directly promotes hair growth via the stimulation of DP and epithelial cells [9
]. Minoxidil stimulated mouse vibrissae follicles in organ culture and induced proliferation of hair epithelial cells near the follicle base [9
]. Further, minoxidil and its derivatives showed cytoprotective activity in vivo and increased prostaglandin E2 (PGE2) production by human DP fibroblasts [11
]. In cultured DP cells, minoxidil-induced hair growth was mediated by adenosine receptors [12
]. Minoxidil also promoted the survival of human DP cells by activating both the ERK and protein kinase B (Akt) pathways, and prevented apoptotic cell death by increasing the ratio of Bcl-2/Bax [15
]. Moreover, minoxidil activated the β-catenin pathway in human DP cells, suggesting a possible mechanism for its anagen prolongation effect [13
]. Minoxidil suppressed androgen receptor (AR)-mediated functions by decreasing AR transcriptional activity in reporter assays and reducing expression of AR targets at the protein level [16
]. Otomo summarized the primary mechanisms of minoxidil action as (a) induction of growth factors in DP cells, such as VEGF, hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1); (b) inhibition of TGF-β-induced apoptosis of hair matrix cells; and, (c) increase of blood flow by dilating hair follicle arteries [14
]. However, there is little evidence that minoxidil can indirectly promote hair growth via ASCs, even though ASCs contribute to the stem cell niche for hair follicles and exert stimulatory effects on hair cycle progression. Therefore, in the present study, we examined possible indirect hair growth-promoting effects of minoxidil via ASCs. Specifically, we investigated whether minoxidil stimulates growth factor secretion by ASCs to enhance follicular cell activity and hair growth.
Minoxidil promotes hair growth directly by stimulating DP and epithelial cells, but previous studies provided little or no evidence for indirect hair growth-promoting effects through the stimulation of ASCs. Therefore, we investigated whether minoxidil stimulates ASCs and enhances hair growth through growth factor release. We first demonstrated that subcutaneous injection of minoxidil-treated ASCs accelerated telogen-to-anagen transition in C3H/HeJ mice and increased hair weight after two weeks. Although minoxidil did not alter the proliferation of ASCs, it did increase migration, tube formation, and secretion of growth factors, notably CXCL1, PD-ECGF, or PDGF-C (the later two through ERK activation). Further, each of these growth factors enhanced DP cell proliferation in vitro and anagen induction in mice, while CXCL1 and PDGF-C also increased the length of isolated mouse vibrissal hair follicles in organ culture. Moreover, these hair growth-promoting effects were synergistic with 2% minoxidil. Collectively, these results indicate that minoxidil stimulates hair growth in part through stimulation of CXCL1, PD-ECGF, or PDGF-C release from ASCs (Figure 7
Chemokine (C-X-C motif) ligand 1 is a secreted growth factor that signals through the G-protein coupled CXC receptor 2, while CXCL1 is known to act as a potent chemoattractant for neutrophils during inflammation [17
]. However, it has not been linked to hair regeneration. PD-ECGF promotes angiogenesis in vivo and stimulates the in vitro growth of multiple endothelial cell types [20
]. It has a highly restricted target cell specificity, acting only on endothelial cells. Of interest, PD-ECGF
was one of the top three genes that were upregulated in ASCs by minoxidil according to qPCR arrays. Moreover, our work in vitro and in vivo revealed that minoxidil-induced PD-ECGF release from ASCs, resulting in DP proliferation and hair growth. On the contrary, it has been well-known that PDGF signaling in the dermis and in dermal condensates is dispensable for hair follicle induction and formation [23
]. For example, PDGF-A secreted from ASCs regulates follicular stem cell activity [2
]. PDGF-A and -B are involved in the induction and maintenance of the anagen phase in the mouse hair cycle [24
]. Our work revealed that PDGF-C also functions in hair growth by upregulating DP proliferation. Although these results might be expected because many growth factors are already known to promote hair cycling, upregulation by minoxidil is meaningful for further usability of this agent to treat androgenetic alopecia. Indeed, the co-application of PDGF-C, CXCL1, or PD-ECGF with 2% minoxidil synergistically increased hair growth (Figure 6
It has been reported that PDGF-D treatment induces growth factor secretion from ASCs via phospho-activation of ERK [5
]. Meldrum’s group also reported that preconditioning (i.e., by hypoxia or transforming growth factor) induced the secretion of numerous growth factors from mesenchymal stem cells through the MAPK pathways [25
]. Similarly, our work revealed that minoxidil induced PD-ECGF and PDGF-C release from ASCs via ERK phosphorylation (Figure 4
). Therefore, it is reasonable to assume that growth factor secretion by ASCs is primarily regulated by the MAPK pathway. However, it appears that CXCL1 is induced via an ERK-independent pathway, suggesting that minoxidil may activate multiple signaling pathways in ASCs. Further investigation is required to identify the pathway mediating minoxidil-induced CXCL1 release.
Minoxidil reportedly increased the proliferation of DP cells in vitro. For example, minoxidil promoted the survival of human DP cells by activating both ERK and Akt pathways, and prevented cell death by increasing the ratio of Bcl-2/Bax [15
]. In addition, minoxidil plus all-trans retinoic acid (ATRA) additively promoted hair growth in human hair follicle culture [29
]. A combination of minoxidil with ATRA elevated phosphorylated ERK, phosphorylated Akt in DP cells and keratinocytes [29
]. However, minoxidil did not increase the proliferation in ASCs, which indicates that proliferative effect of minoxidil is cell type dependent.
ACSs are considered an important component of other stem cell niches. Indeed, it has been shown that adipocyte lineage cells are part of the skin stem cell niche that drives hair cycling, and it was suggested that PDGF-A expression by immature adipocytes is a key regulator of follicular stem cell activity [2
]. Although we did not investigate the effects of minoxidil or ASC-released growth factors on DP stem cells, ASC-released growth factors (specifically CXCL1, PD-ECGF and PDGF-C) induced DP cell proliferation, consistent with a pivotal role in the regulation of DP stem cells. To determine whether the effect of minoxidil on specific to the ASCs, minoxidil didn’t induce cell growth either migration of dermal fibroblast cells (Figure S1
We previously reported that preconditioned ASCs by growth factor, such as PDGF-D, enhance hair generative potential in tellogen to anagen induction model [5
]. The purpose of this model is to induce anagen phase more quickly after we injected ASCs by paracrine effect. Anagen hair induction was not limited to the injection site. Instead, darkening of the skin or hair appeared across all areas of the back. We showed increased hair weight in ASCMXD
-treated mice, which indicates the increase of hair length as well as hair number. The measurement of hair weight as an evaluation for hair growth is not enough to conclude increased telogen-to-anagen induction. Therefore, we examined hair follicle and proliferating DP cells in sectioned skin. The skin section of ASCMXD
-treated mice showed higher number of mature hair follicle compared to vehicle- or ASCCtrl
-treated mice (Figure 1
C). In addition, most hair follicles of ASCMXD
-treated mice showed DP with Ki67+
cells contrary to vehicle- or ASCCtrl
-treated mice (Figure 1
D). This result suggests that minoxidil can promote telogen to anagen induction, thereby promoting hair growth. Although the measurement of hair weight is not enough to examine the growth of hair follicle and DP proliferation, this method is easy to monitor hair growth quickly.
In summary, subcutaneous injection of minoxidil-treated ASCs accelerated the telogen-to-anagen transition in mice, and direct minoxidil treatment increased migration, tube formation, and growth factor secretion by ASCs. The most strongly upregulated growth factors, CXCL1, PD-ECGF and PDGF-C, individually enhanced anagen induction in mice, while CXCL1 and PDGF-C also increased the length of isolated mouse vibrissal hair follicles in organ culture. Therefore, minoxidil can be used as a novel ASC preconditioning agent for hair regeneration.
4. Materials and Methods
4.1. Cell Culture
Human adipocyte-derived stem cells (ASCs) were isolated via liposuction of subcutaneous fat as described previously [30
]. Briefly, fat was washed with phosphate-buffered saline (
PBS), added 0.075% collagenase and incubated for 45 min at 37 °C with gently shaking. The pellet after centrifugation was filtered with 100 µm nylon mesh, gathered again after centrifugation. Then, cells were cultured with essential medium, including α-minimum essential media (MEM) (Hyclone, Logan, UT, USA), 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% anti-antibiotics (Gibco) until passage 3. Then, medium was changed to minimum essential medium including α-MEM, 10% fetal bovine serum, and 1% penicillin/streptomycin (Gibco) at four passages. ASCs were used at passages 5–7 for all of the experiments. Characterization of ASCs was performed using flow cytometry. ASCs were positive for CD44, CD73, CD90, CD105, human leukocyte antigen-I (HLA-I), and podocalyxin (PODXL), but were negative for hematopoietic markers such as CD34 and CD45 [32
]. Multipotent differentiation potential was examined as described previously [34
], and ASCs could be differentiated into adipocytes, osteocytes, and chondrocytes. We purchased human DP cells from PromoCell (#C-12071, PromoCell, Logan, Heidelberg, Germany). Human DP cells were cultured in Follicle DP Cell Medium (C-26505; PromoCell) with SupplementMix (C-39625; PromoCell) and 0.1% anti-antibiotics (Gibco). DP cells were used at passages 3, 4. We also purchased human dermal fibroblast cells from PromoCell (#C-12302). Dermal fibroblasts were cultured in dulbecco modified eagle medium (DMEM)/high glucose (Hyclone) with 10% fetal bovine serum and 1% penicillin/streptomycin, and were used at passages 8–10. All of the cells were maintained at 37 °C in a humidified 5% CO2
incubator (Theremo Fisher scientific, Waltham, MA, USA).
4.2. Cell Growth Assay
For measuring the effects of minoxidil on ASC proliferation, cells were seeded in 6-well plates at 1 × 104
cells/well, treated with minoxidil (0.1–100 µM), and incubated for two days in an InCu-saFe CO2
incubator (Panasonic, Kadoma, Osaka, Japan). Live cell number was determined using the IncuCyte zoom2014A live cell analysis system (Panasonic, Kadoma, Osaka, Japan) [5
]. Alternatively, ASCs were seeded in 12-well plates at 5000 cells/well, treated with minoxidil (20, 50, or 100 µM), and incubated for seven days. Cells were then trypsinized, stained with Trypan Blue (Sigma-Aldrich, St. Louis, MO, USA), and counted each day using a hemocytometer hemocytometer. To assess the effects of growth factors secreted by ASCs on DP cell proliferation, 1 × 104
DP cells were seeded in 12-well plates and treated with the indicated synthesized peptides for up to three days. Cells were trypsinized and stained with Trypan Blue. Viable cell number was counted each day using a hemocytometer.
4.3. Scratch Migration Assay
Cells were seeded into 6-well plates and cultured to confluence. A sterile 1 mL pipette tip was used to scratch the cell monolayer. Cultures were then washed with PBS to removed deplated cells and cultured with minoxidil in serum-free medium for four days. Migration of cells into the scratched area (wound closure) was visualized using a ZEISS Observer D1 microscope (Carl ZEISS, Oberkochen, Germany). Multiple images were acquired per well and average cell counts within the wound monitored over 4 days.
4.4. Cell Migration Assay Using Transwell
ASCs (1 × 104/well) were suspended in serum-free medium and seeded on the upper side of transwell membrane plates (BD falcon, BD Biosciences, San Jose, CA, USA). After 2 h, minoxidil in serum-free medium were introduced into the lower chambers. Cultures were incubated for two days to allow transwell migration. Inserts were then removed and the upper surface was cleaned of non-migrating cells using cotton swabs and washed with PBS. The inserts were stained with 0.1% formalin/10% crystal-violet solution (Sigma-Aldrich) and cell number analyzed under a ZEISS Observer D1 microscope. Multiple images (15–20) per insert were acquired, and average cell counts were calculated.
4.5. Tube Formation Assay Using Matrigel
Twelve-well plates were coated with matrigel (BD matrigel matrix, BD Biosciences) and were dried for 2 h at 37 °C. ASCs in endothelial cell basal medium-2 (EBM-2, LONZA, Walkersville, MD, USA) plus minoxidil were plated on matrigel-coated wells and incubated for 12–16 h at 37 °C. The number of tubes was analyzed under a ZEISS Observer D1 microscope [3
], and the expression level of endothelial cell markers was analyzed by qPCR.
4.6. RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from ASCs using Trizol reagent (Invitrogen, Carlsbad, NY, USA) and subjected to cDNA synthesis using oligodT and the HelixCript™ Thermo Reverse Transcription System (Nanohelix, Madison, WI, USA), according to the manufacturer’s instructions. BrightGreen qPCR master mix-ROX (ABM, New York, NY, USA) was used for PCR reactions.
4.7. Western Blot
For western blot of phospho-ERK, minoxidil and PD98059 (Sigma-Aldrich) was treated for 30 min–1h. Cells were lysed with protein extraction solution (PRO-PREPTM
; iNtRON, Seoul, Korea) containing phosphatase inhibitor (Na3
; Roche, Pleasanton, CA, USA). Western blot analysis was performed as described previously [8
] using the following primary antibodies: rabbit anti-p42/44 (1:1500; Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-p42/44 (1:1500; Cell Signaling Technology), and mouse anti-α-tubulin (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA). Western blot images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Science, Pittsburgh, PA, USA).
4.8. Animal Experiment
Mice were maintained and anesthetized according to a protocol that was approved by the US Pharmacopoeia and the Institutional Animal Care and Use Committee of Yonsei University (IACUC120002, 17 July 2015). The dorsal area of 6.5-week-old C3H/HeN mice in the telogen stage of the hair cycle was shaved with a clipper and electric shaver, with special care taken to avoid damaging the bare skin. Naïve ASCs or minoxidil-treated ASCs were only once injected into the dorsal skin of shaved mice. The growth factors (100 ng/mL per one day) were injected into the dorsal skin of shaved mice every day for 12 days. CXCL1 (R and D Systems, Minneapolis, MN, USA), PD-ECGF (R and D Systems) and PDGF-C (PeproTech, Rocky Hill, NJ, USA) proteins were purchased from each companies. For application with 2% minoxidil, CXCL1, PD-ECGF or PDGF-C (1 µg/mL) with 2% minoxidil (Rogaine; Johnson and Johnson Healthcare Products, Skillman, NJ, USA) were applied on the dorsal skin of shaved mice. Any darkening of the skin (indicative of hair cycle induction) was carefully monitored by photography. After 14 days, the dorsal hair was shaved and weighed to estimate growth rate [3
4.9. Vibrissae Follicle Organ Culture
For organ culture of vibrissae hair follicle, the vibrissae hair follicle was cut from C3H/HeN mice, washed with PBS and cultured in defined medium (Williams E medium supplemented with 2 mM l-glutamine, 10 µg/mL insulin, 10 ng/mL hydrocortisone, 100 U/mL penicillin, and 100 µg/mL streptomycin, without serum) including CXCL1 (5 ng), PD-ECGF (20 ng) and PDGF-C (20 ng) for three days.
4.10. BrdU Labeling
For BrdU labeling assay, 4 × 104 cells were seeded in six well plates and incubated for 3 days after adding three proteins. BrdU (Sigma-Aldrich) was added in cell culture media to a final concentration of 200 mM and incubated for 4 h at 37 °C with 5% CO2. The cells were fixed with 4% paraformaldehyde, incubated with mouse anti-BrdU (1:500) (Abcam, Iowa City, IA, USA) overnight at 4 °C, and then incubated with secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (Invitrogen), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Immunofluorescence staining was imaged using ZEISS LSM700 confocal microscope (Carl ZEISS, Oberkochen, Germany). For calculation of the percentage of BrdU+ cells, we counted the number of BrdU+ cells and DAPI+ cells (all cells) in every same size pictures using Adobe photoshop CS6 extended program (Yonsei University, Seoul, Korea), and calculated the percentage of BrdU+ cells.
4.11. Hematoxylin/Eosin and Immunofluorescence Staining
For Hematoxylin and eosin staining, paraffin sections were dewaxed using xylene for 30 min, hydrated in 100%-, 90%-, 80%- and 70% EtOH and was dipped into Mayer’s hematoxylin (Sigma-Aldrich) for 8 min, and then rinsed in water for 1 minute. Slide was dipped again into eosin Y (Sigma-Aldrich) for 80 s, dehydrated with 70%-, 80%-, 90%- and 100% EtOH, washed with fresh xylene for 30, dried, and mounted with mounting medium. Immunofluorescence staining was performed using standard protocols. Briefly, paraffin sections were dewaxed using xylene for 30 min, hydrated in 100%-, 90%-, 80%- and 70% EtOH and antigen retrieval was performed by boiling using microwave in antigen retrieval buffer (Dako, Carpinteria, CA, USA) for 2 min. The sections were treated with rabbit Ki67 antibody (1:300) (abcam) overnight at 4 °C, and were then incubated with secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Immunofluorescence staining was imaged using ZEISS LSM700 confocal microscope.
4.12. Statistical Analysis
All of the experiments were performed more than three times with independent cultures. Data are presented as mean ± standard error (SEM). Means were compared by Student’s t-test. For all statistical tests, a 0.05 level of confidence was accepted as statistically significant.