Exosomes Derived from Dermal Papilla Cells Mediate Hair Follicle Stem Cell Proliferation through the Wnt3a/β-Catenin Signaling Pathway

Both hair follicle stem cells (HFSC) and dermal papilla cells (DPC) are essential for hair follicle growth and proliferation. In this study, HFSCs and DPCs that made signature proteins like KRT14, KRT15, KRT19, α-SMA, and Versican were obtained. Cell coculture systems between HFSCs and DPCs were used to measure the increased PCNA protein content in HFSCs. Additionally, exosomes from dermal papilla cells (DPC-Exos), the overexpression and silencing of Wnt3a, could regulate the Wnt/β-catenin signaling pathway downstream genes. After collecting DPC-ExosOE-Wnt3a, the treatment of HFSC with DPC-ExosOE-Wnt3a showed that DPC-ExosOE-Wnt3a could upregulate the mRNA expression of downstream genes in the Wnt/β-catenin signaling pathway and that DPC-ExosOE-Wnt3a enhanced the proliferation of HFSCs while inhibiting their apoptosis. These findings suggest that DPC-Exos could regulate HFSC cell proliferation via the Wnt3a/β-catenin signaling pathway. This research offers novel concepts for the molecular breeding and efficient production of Angora rabbits, as well as for the treatment of human hair problems.


Background
The development and growth of hair follicles is a complex process. Several cells, such as HFSC and DPC, are involved in the biological processes. Cell-cell interactions are essential to the formation of tissues and the performance of physiological processes in multicellular organisms [1]. Hair follicles (HFs), which contain both dermal and epidermal components, are a unique organ in mammals [2]. The HFSC and DPC are the two most significant cells in HFs. HFSCs are detected in the follicle's upper portion [3]. Stem cells can generate the interfollicular epidermis, hair follicle components, and sebaceous glands. In a simulated in vivo environment, the protruding epithelial stem cells can create a new hair follicle [4]. The DPCs are located at the base of the HF. A drop in the DPC number, which specifies hair size, form, and cycling, is the primary cause of HF decline [5].
DPCs and HFSCs communicate with one another. On the one hand, HFSC can be in vitro activated by the presence of DPCs [6]. HFSC, on the other hand, regenerates the dermal papilla [7]. Extracellular vesicles (EVs) have been regarded as reliable intercellular messengers. This is because of their ability to transport proteins, lipids, and nucleic acids, which influence a multitude of physiological and pathological processes in both parent and recipient cells [8]. Exosomes are endosomalderived EVs with sizes between 40 and 160 nm [9]. Exosomes include a variety of cellular components such as DNA, micro-RNAs, mRNA, RNA, lipids, metabolites, and cytosolic and cell-surface proteins. DPC-Exos (exosomes made from dermal papilla cells) improve the capacity of cultured dermal papilla spheres to stimulate hair development [10].
One of the most crucial and significant signals for controlling the formation of hair follicles is the Wnt signaling pathway [11,12]. Wnt signaling has an impact on both the structure of the hair shaft and the control of hair growth [13]. Several Wnt family members, including Wnt3, Wnt3a, Wnt10a, and Wnt10b, are associated with HF morphogenesis in the developing skin [14,15]. Wnt3a can play the role of inductive signals, which helps the DP remain in an anagen state [16]. Wnt3a signaling, which primarily employs the traditional route with β-catenin as the main protein, is critical for controlling DPC aggregation [17].
The exosomal Wnt3a from a HERS cell line was used to increase the migration, proliferation, and differentiation of dental papilla cells into odontogenic cells as well as the activation of Wnt/β-catenin signaling [18]. Wnt3a and β-catenin levels may be increased by oxidized-sodium-alginate-(OSA-) encapsulated EVs, which promote hair growth [19]. The current study discovered that DPC-Exos, such as DPC-Exos miR-181a-5p , which regulates HF growth and development and promotes the Wnt/β-catenin signaling pathway [20], and DPC-Exos miR-22-5p , which mediates HFSC proliferation and differentiation [21], play critical roles in HF growth. However, the precise mechanism by which DPC-Exos regulate HFSC quiescence and activation is unknown. In this study, we focus on coculturing DPC and HFSC to investigate how DPC-Exos regulate HFSC proliferation via the Wnt3a/β-catenin signaling pathway.

Isolation and Identification of the DPCs and HFSCs.
DPCs were isolated using a combination of microdissection and two-step enzymatic digestion, while HFSCs were isolated using only the two-step enzymatic digestion. We obtained pure HFSCs by combining differential digestion and the type IV collagen fast adhesion technique. Primary DPCs spread slowly and had fibroblast-like attributes (Figure 1(a)). The HFSCs had an egg-like form and were spherical. According to immunofluorescence labeling, KRT14, KRT15, and KRT19 proteins were preferentially expressed in HFSCs, while α-SMA and Versican proteins were exclusively expressed in DPCs ( Figure 1).

DPCs Promoted the HFSC Proliferation in Cell
Cocultured System. Transwell plates were used to establish the cocultured system of DPCs and HFSCs. DPCs were attached to the upper chamber, whereas HFSCs were located at the bottom. After a 48 h coculture, indirect immunofluorescence labeling was used to identify PCNA expression in HFSCs. The findings demonstrated that HFSCs had a much higher level of PCNA protein expression ( Figure 2). qRT-PCR showed that the expression of the mRNA for Wnt3a, LEF1, ALPL, and IGF1 was greatly increased in the HFSC with the coculture system.

Wnt3a Enhances Proliferation and Inhibits Apoptosis of
HFSCs by Wnt/β-Catenin Signaling Pathway. Anagen gene expression in DPCs has been reported to be maintained by Wnt3a [22,23]. The Wnt3a mRNA expression in the HFSC could be significantly knocked down and overexpressed using siRNA-Wnt3a and pcDNA3.1-Wnt3a, respectively (Figures 3(a) and 3(b)). Additionally, the knockdown of Wnt3a significantly reduced the mRNA expression of downstream genes of the Wnt/β-catenin signaling pathway, including CTNNB1 and LEF1 (Figure 3(b)). Likewise, the overexpression of Wnt3a resulted in a rise in the expression of these genes (Figure 3(d)).
DPC-Exos OE-Wnt3a had the largest average particle size and concentration (Figure 4(b) and Table 1). Following that, the exosome-specific proteins CD9, TSG101, and Alix were significantly expressed in the DPC-Exos (Figure 4(c) and Figure S1). In the coculture system, DPC-Exos were labeled with DiI and added to HFCSs for 24 h; fluorescence analysis revealed that DiI-DPC-Exos could enter HFSCs in the Transwell plate ( Figure 4(d)). Furthermore, qRT-PCR results revealed that DPC-Exos OE-Wnt3a significantly increased the mRNA expression of the Wnt/β-catenin signaling pathway downstream genes such as WNT3a, LEF1, and CTNNB1 after feeding HFSCs DPC-Exos (P < 0:01) ( Figure 5    Oxidative Medicine and Cellular Longevity study, and they were identified as KRT14, KRT15, KRT19, α-SMA, and Versican, which is consistent with earlier studies [27][28][29]. Coculture systems are essential in all studies of cellcell interactions and have long been used to investigate interactions between cell populations [30]. To study PD-L1/PD-1 interactions in the tumor microenvironment in vitro, mouse-derived gastric cancer organoids and autologous immune cells were cocultured [31]. After activating the androgen and Wnt/β-catenin signaling pathways, HFSC differentiation was assessed in a coculture model with DPC or culturing with DPC-conditioned media [32]. PCNA (proliferating cell nuclear antigen) has been shown as one of the proliferation markers [33]. PCNA was used to detect cell proliferation in HF [34,35]. The HFs in combined treatment of IGF-1 and EGF groups showed higher PCNA expression than the control group [36]. We found that coculturing HFSCs with DPC led to a higher level of PCNA protein, demonstrating that DPCs promoted the HFSC proliferation in cell cocultured system.
Exosomes from different cell types frequently contain distinct RNA and protein payloads that reflect the phenotypes of their parental cells [37], and they may also contain cell or tissue-specific markers that can be used to identify the origin of an exosome [38]. Many pathophysiological conditions, including cancer, immune responses, cardiovascular diseases, regeneration, and stem cell-based therapies, have been linked to EV activity [8]. An emerging area of study is the potential roles and applications of EVs in HF function [39]. DPC proliferation is promoted by neural progenitor cell-derived miR-100 [40]. Milk-exo accelerates the hair cycle transition from telogen to anagen phase by activating the Wnt/β-catenin pathway [41]. Many studies have revealed that DPC-Exos can mediate outer root sheath cell proliferation and migration [42], as well as hair follicle stem  Wnt3a   8000  7000  6000  5000  4000  3000  5  4  3  2       Oxidative Medicine and Cellular Longevity cell proliferation and differentiation [21]. We used exosomes collected after plasmid transfection as a tool for content targeting and delivery into recipient cells (HFSCs). The EVs are with sizes between 40 and 160 nm [8,9], in our study. The average particle size of the DPC-Exos is around 80 nm, which is consistent with exosome size.

Conclusions
The study found that DPCs aided HFSC proliferation in a cell coculture system. Wnt3a promotes HFSC proliferation and inhibits apoptosis via the Wnt/β-catenin signaling pathway. DPC-Exos may regulate HFSC cell proliferation via the Wnt3a/β-catenin signaling pathway. This research provides new ideas for molecular breeding and efficient production of Angora rabbits, as well as the treatment of human hair diseases.

Primary
Culture of Rabbit Vibrissae DPC. All experiments with the rabbits were approved by the Animal Care and Use Committee of Yangzhou University (Yangzhou, China, 18 March 2021, No. 20210318). Angora rabbits (aged 6 months) were purchased from the Jiangdu Renyuan Rabbit Industry. The DPCs were isolated through a combination of microdissection and two-step enzymatic digestion [50]. The collected vibrissae were washed with PBS, and soaked in 75% alcohol for 1 min. The 0.25% Dispase II (Sigma) was used to isolate the epidermic cells (4°C, overnight). The next morning, the HFs were isolated by stereoscopic microscope. The bulb of the HF was cut with ophthalmic scissors and digested with 0.2% collagenase II (ThermoFisher) at 37°C for few hours. Dermal papilla tissue was obtained by centrifugation and cultured in cell incubator. After a few days, the DPCs crawled out of the dermal papilla and grew adherently in the cell culture dish. The cells were passaged in 0.25% trypsin and cultured with mesenchymal stem cell medium (MSCM) complete medium (ScienCell, USA).

HFSC Isolation and Growth Conditions.
We used ophthalmic scissors to cut the Vibrissa of the Angora rabbit and placed them in icy PBS. Skin dissected from rabbit vibrissae was washed thrice with PBS and soaked it in 75% alcohol for 5 min. After washing thrice with PBS, the skin was cut into 2 × 2 mm long strips and incubated in Dispase II (Sigma-Aldrich) overnight at 4°C. The epidermis was peeled off in the clean bench, leaving the dermis intact. The skin tissue fragments were incubated in 0.25% type IV collagenase for 2 h at 37°C. An equal amount of protease inhibitor was added to stop digestion and pipetted to mix uniformly, followed by filtration through a 200-mesh sieve, collection of the filtrate, and centrifugation at 1000 rpm for 10 min. The supernatant was discarded, and the medium containing DMEM/F12 was added to resuspend the cells and placed them in a 35 mm petri dish. The petri dish was incubated at 37°C under 5% CO 2 atmosphere and cultured. The medium was replaced after every 2 days.

Cocultivation of DPCs and HFSCs.
The coculture system of DPCs and HFSCs was established according to our system used before [20]. For detail, the Transwell (Cat#:  3412   5.11. Exosome Extraction. According to the instructions of the Total Exosome Isolation Kit (from cell culture medium) (lot: 4478359, Invitrogen), DPC cell culture medium was centrifuged at 2000 ×g for 30 min. The supernatant was transferred to a new tube and treated with 0.5 times the supernatant volume of the reagent, mixed well, and incubated overnight at 2-8°C. The next day, the mixed liquid was centrifuged at 10,000 ×g for 1 h at 4°C. The exosomes remained at the bottom.
5.12. Transmission Electron Microscopy. The exosomes (5 μL) were diluted to 10 μL, and 10 μL of the sample was drawn and dropped on the copper mesh for 1 min, followed by removal of the floating liquid with a filter paper. Then, 10 μL of phosphotungstic acid was added dropwise to the copper mesh for 1 min, and the suspension was removed through the filter paper and dried at room temperature for a few minutes. Finally, they were imaged under 80 kV electron microscopy.
5.13. Nanoparticle Tracking Analysis. The exosomes (5 μL) were removed and diluted to 30 μL, and the particle size and concentration of exosomes were detected by the NanoFCM instrument (Flow NanoAnalyzer).
5.14. Statistical Analysis. SPSS 22.0 was used for data analysis. Paired samples T tests were conducted for relative expression analysis. The graphical representations were performed using the GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). P < 0:05 was considered to indicate statistically significant difference, while P < 0:01 was considered to indicate extremely significantly difference.

Ethical Approval
Animal experiments were strictly carried out in accordance with the recommendations of the Yangzhou University Animal Care and Use Committee.

Conflicts of Interest
The authors declare that they have no competing interests.