Effects of oxytocin on the hair growth ability of dermal papilla cells

Oxytocin (OXT) is a neuropeptide hormone termed “love hormone” produced and released during childbirth and lactation. It is also produced in response to skin stimulation (e.g., during hugging and massaging) and music therapy. The effects of OXT on various organs have been revealed in recent years; however, the relationship between hair follicles and OXT remains unclear. In this study, we examined the effects of OXT on dermal papilla (DP) cells that control hair growth by secreting growth/regression signals. Gene expression analysis revealed that DP signature markers were significantly upregulated in DP cells treated with OXT. In addition, we tested the hair growth-promoting effects of OXT using in vitro hair follicle organoids. OXT promoted the growth of hair peg-like sprouting by upregulating the expression of growth-promoting factors, including genes encoding vascular endothelial growth factor A (VEGFA). This study highlights the positive effects of OXT in hair follicles and may assist in the development of new treatments for alopecia.

the hair growth-promoting ability of OXT was investigated using an in vitro hair growth model.The findings of this study may help in the development of new treatment strategies for hair loss.
We further investigated the key signaling pathways activated by OXT stimulation.DP cells were treated with/ without 10 µM OXT for 6 days, and the gene expressions were investigated through comprehensive RNA-seq analysis.RNA-seq results revealed 18,929 DEGs between 0 and 10 µM OXT, of which 8480 genes were upregulated in 10 µM OXT-treated DP.The top 10 enriched pathways represented by these upregulated genes included the cytokine-cytokine receptor interaction and OXT signaling pathway (Fig. 5a).In addition, genes associated with hair growth-promoting factors, including VEGFA, FGF7, and BMP2, were also upregulated by 10 µM OXT treatment (Fig. 5b).These results suggest that mechanisms associated with DP cell activations include cytokine and growth factor secretion through the activation of OXT signaling pathway.Minoxidil, a commercially available Hair growth assay using an in vitro hair growth model.We recently developed an in vitro hair follicle organoid model (termed the hair follicloid) to identify hair growth-promoting factors 36 .In our previous study, hair follicloids were prepared by culturing human DP and epithelial cells in a medium supplemented with a low concentration of Matrigel.Matrigel significantly enhanced the self-organization capabilities of epithelial and mesenchymal cells, resulting in spherical aggregation and subsequent hair peg-like sprouting.Peg-like hair sprouting is composed of hair cortex cell marker AE13 positive cells, but the structure is immature compared with native hair follicles.However, the hair follicloid was sufficient to elongate the peg-like hair sprouting in response to minoxidil.In the present study, we used hair follicloids to examine the effect of OXT on hair growth.Human DP and epithelial cells were suspended in a medium supplemented with 2 v/v% Matrigel and cultured in 96 well spheroid formation plates to prepare hair follicloids.Hair follicloids were treated with 0 and 10 µM OXT after day 4, and hair peg-like sprouting was measured from day 4 to 10 (Fig. 6a).Hair peg-like sprouting was elongated both in 0 and 10 µM OXT-treated hair follicloids.The sprouting length was significantly longer in the 10 µM OXT-treated group than in the untreated control group on days 8 and 10 (Fig. 6b,c).Next, we www.nature.com/scientificreports/examined the expression of DP-produced growth factors, including VEGFA and PDGFB.VEGFA and PDGFB were significantly upregulated in the presence of OXT (Fig. 6d), suggesting that the elongation of hair peg-like sprouts was promoted by the production of growth factors.These results indicate that OXT has the potential to be a hair growth reagent, although further studies are needed to determine their effects under in vivo environments of hair follicles.

Discussion
A recent study has shown that OXT accumulates in hair shafts and can be evaluated as a biomarker of stress 37 ; however, to the best of our knowledge, there are no studies on the effects of OXT on cells in hair follicles.We found that OXT stimulates DP cells to promote the secretion of growth factors.This finding inspired us to apply OXT therapy to the hair loss treatment, and the hair growth-promoting effects of OXT were confirmed using our in vitro drug screening model.This study revealed the roles of OXT in hair follicles in in vitro, but it would be crucial to understand whether OXT affects hair follicles in more complex in vivo environments for appreciation as a hair loss treatment drug.OXT has been shown to act on multiple cell types in addition to hair follicles 24,25 , and its use as a treatment for alopecia requires consideration of its side effects on multiple organs.The future study will investigate the understanding of hair growth and side effects using an alopecia model mouse.Also, the present study used DP cells derived from healthy donors, and further investigations must be conducted using cells from patients with alopecia.In addition, understanding age-and sex-dependent efficacy is necessary because OXT production differs with age and gender.OXT concentration and treatment time should be more precisely optimized through these experiments.By advancing these studies, we would like to verify whether OXT can be a new drug for the treatment of alopecia.The transcriptome analysis of the DP cells revealed that OXT treatment promoted OXT signal transduction and cytokine/growth factor secretion.The DP-secreted factors promoted the growth of hair peg-like sprouting in hair follicloids.However, there are many possible mechanisms by which OXT promotes hair growth.It should be noted that epithelial cells in hair follicloids also have OXTR (Supplementary Fig. 5).Further investigation is needed to understand whether OXT acts directly on epithelial cells to stimulate cell proliferation and growth of hair peg-like sprouting.
Although we investigated the effect of OXT as a hair growth-promoting drug, it may also be used to restore DP cell function in hair regeneration medicine.When DP cells are transplanted with epithelial cells into the skin, DP cells can induce de novo hair follicle regeneration in the recipient's skin 38 .However, the hair regeneration ability of DP cells is gradually lost during expansion culture 39 .We previously developed approaches to restore the hair regeneration ability of DP cells using electrical stimulation and gel bead culturing [40][41][42] .Combining these methods with OXT may further improve the hair regeneration efficiency of expanded DP cells, which will be examined in future studies.
In conclusion, we showed that OXT activated the DP cells to promote growth factor secretion for hair growth.These findings encourage further investigation for clinical applications of OXT therapy in patients suffering from hair loss.

Methods
Preparation of human DP and epithelial cells.Adult human DP cells were obtained from PromoCell (Heidelberg, Germany), passaged up to passage four with R-STEM in hMSC high-growth medium (EM1; Roto, Japan), and used for 2D culture.Adult human follicular keratinocytes (epithelial cells) were obtained from Sci-enCell Research Laboratories (Carlsbad, CA, USA).DP cells at passage four and epithelial cells at passage one were used for organoid culture.Incubator gas tension was maintained at 21% O 2 and 5% CO 2 at 37 °C.
OXT treatment of DP cells.DP cells (2 × 10 4 cells) were suspended in 0.5 mL EM1 medium supplemented with 0, 0.1, 1, or 10 μM OXT (Peptide Institute Inc., Japan) and seeded into the wells of a 24-well cell culture plate (Corning Inc., Corning, NY, USA).The culture medium was replaced with a fresh medium every 3 days.The cells were counted using a cell counter (Chemometec, Denmark) after 3 min of trypsin-EDTA treatment.Gene expression in DP cells was assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR) after 6 days of culture.

Gene expression analysis.
Total RNA was extracted from the samples using RNeasy Mini Kit (Qiagen, Hilden, Germany) and used for complementary DNA synthesis using the ReverTra Ace ® RT-qPCR Kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.Subsequent qRT-PCRs were performed using the StepOne Plus RT-PCR system (Applied Biosystems, Waltham, MA, USA) with SYBR ® Premix Ex Taq™ II (Takara Bio, Kusatsu, Japan) and primers for amplifying human ALP, VCAN, LEF1, WNT5A, BMP4, NOG, VEGFA, PDGFB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1).All primers used in this study are listed in Table S1.All gene expression levels were normalized to that of GAPDH.The 2 −∆∆Ct method was used to determine relative gene expression levels, and they were presented as the mean ± standard error of three independent experiments.Statistical evaluation of numerical variables was conducted using Tukey's or Student's t-test, where a p-value of < 0.05 indicated statistical significance.The membranes were washed with TBST solution three times, then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000 dilution, Cell Signaling Technology) at room temperature for 1 h.Protein bands on the membrane were visualized using ECL Prime (GE Healthcare, Buckinghamshire, UK) and an Amersham Imager 600 RGB (Cytiva, Tokyo, Japan).The relative protein levels were evaluated using the GAPDH expression level as a reference.
RNA-seq analysis.Total RNA was extracted using RNeasy Mini Kit (Qiagen) from DP cells with/without 10 μM OXT treatment for 6 days.RNA-seq analysis was performed by Takara Bio.The significantly upregulated genes in DP cells subjected to OXT treatment were used for the Kyoto Encyclopedia of Genes and Genomes pathway analysis with the Database for Annotation, Visualization, and Integrated Discovery (http:// david.abcc.ncifc rf.gov/) [44][45][46] .

Statistical analysis.
Statistical analyses of gene expression levels and the length of hair sprouts were conducted using Tukey's test or Student's t-test, and the results were considered statistically significant at p < 0.05.All data are presented as mean ± standard error.

Figure 1 .Figure 2 .
Figure 1.Scheme outlining the identification of oxytocin (OXT) effects on dermal papilla (DP) cells.DP cells and hair follicloids were supplemented with oxytocin for activation and hair growth promotion.

Figure 3 .Figure 4 .
Figure 3. Proliferation and gene expression analysis of dermal papilla (DP) cells cultured in 2D.(a) Number of expanded cells at 3 and 6 days of culture.(b) Expression of DP signature marker genes.GAPDH was used as a reference gene to normalize expression.Error bars represent the standard error of the mean calculated from three experiments for each condition.Using Tukey's test, the numerical variables were statistically evaluated; * indicates p < 0.05.

Figure 5 .Figure 6 .
Figure 5. Gene chip analysis and enriched signaling pathway inhibition.(a) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of upregulated differentially expressed genes (DEGs) showing the top 10 enriched pathways.(b) Heat map DEGs between DP cells treated with 0 μM and 10 μM oxytocin (OXT).