Fu Y, Han YT, Xie JL, Liu RQ, Zhao B, Zhang XL, Zhang J, Zhang J. Mesenchymal stem cell exosomes enhance the development of hair follicle to ameliorate androgenetic alopecia. World J Stem Cells 2025; 17(3): 102088 [DOI: 10.4252/wjsc.v17.i3.102088]
Corresponding Author of This Article
Jing Zhang, PhD, Professor, Research Center for Translational Medicine at East Hospital, School of Life Science, Tongji University, No. 1239 Siping Road, Shanghai 200092, China. 96755@tongji.edu.cn
Research Domain of This Article
Developmental Biology
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Yu Fu, Yao-Ting Han, Rong-Qi Liu, Bo Zhao, Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai 200092, China
Jun-Ling Xie, Xing-Liao Zhang, Research Center for Translational Medicine at East Hospital, School of Medicine, Tongji University, Shanghai 200092, China
Jun Zhang, Jing Zhang, Tongji Lifeng Institute of Regenerative Medicine, Tongji University, Shanghai 200092, China
Jun Zhang, Research Center for Translational Medicine at East Hospital, Shanghai Institute of Stem Cell Research and Clinical Translation, School of Medicine, Tongji University, Shanghai 200092, China
Jing Zhang, Research Center for Translational Medicine at East Hospital, School of Life Science, Tongji University, Shanghai 200092, China
Co-corresponding authors: Jun Zhang and Jing Zhang.
Author contributions: Zhang J and Zhang J mainly designed and led the process of the project and as co-corresponding authors of this manuscript. Fu Y and Han YT wrote the manuscript and contributed equally to this manuscript as co-first authors of this manuscript. Fu Y, Xie JL, Liu RQ, Zhao B, and Zhang XL performed the experiments; Fu Y, Xie JL, Zhao B, and Zhang XL analyzed the data; Fu Y, Han YT, and Liu RQ performed statistical analysis. All authors read and approved the final manuscript.
Supported by the Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai and the China Postdoctoral Science Foundation, No. 2022M722409.
Institutional review board statement: The study protocol conformed to the Declaration of Helsinki and was approved by the Committee of Ethics on Experimentation of Tongji University (Shanghai, China), approval No. [2024]094.
Institutional animal care and use committee statement: This study was approved by Tongji University, with the ethics approval No. TJAA09524102.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: All data can be supplied for reasonable requests.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Jing Zhang, PhD, Professor, Research Center for Translational Medicine at East Hospital, School of Life Science, Tongji University, No. 1239 Siping Road, Shanghai 200092, China. 96755@tongji.edu.cn
Received: October 9, 2024 Revised: January 21, 2025 Accepted: February 26, 2025 Published online: March 26, 2025 Processing time: 163 Days and 3.5 Hours
Abstract
BACKGROUND
Mesenchymal stem cells (MSCs) and their secretome have significant potential in promoting hair follicle development. However, the effects of MSC therapy have been reported to vary due to their heterogeneous characteristics. Different sources of MSCs or culture systems may cause heterogeneity of exosomes.
AIM
To define the potential of human adipose-derived MSC exosomes (hADSC-Exos) and human umbilical cord-derived MSC exosomes (hUCMSC-Exos) for improving dermal papillary cell proliferation in androgenetic alopecia.
METHODS
We conducted liquid chromatography-mass spectrometry proteomic analysis of hADSC-Exos and hUCMSC-Exos. Liquid chromatography-mass spectrometry suggested that hADSC-Exos were related to metabolism and immunity. Additionally, the hADSC-Exo proteins regulated the cell cycle and other 9 functional groups.
RESULTS
We verified that hADSC-Exos inhibited glycogen synthase kinase-3β expression by activating the Wnt/β-catenin signaling pathway via cell division cycle protein 42, and enhanced dermal papillary cell proliferation and migration. Excess dihydrotestosterone caused androgenetic alopecia by shortening the hair follicle growth phase, but hADSC-Exos reversed these effects.
CONCLUSION
This study indicated that hair development is influenced by hADSC-Exo-mediated cell-to-cell communication via the Wnt/β-catenin pathway.
Core Tip: Liquid chromatography-mass spectrometry proteomic analysis was conducted to reveal the commonalities and heterogeneities across the human adipose-derived mesenchymal stem cell exosomes (hADSC-Exos) and human umbilical cord-derived mesenchymal stem cell exosomes. A total of 232 common proteins were found in hADSC-Exos and were categorized into 10 functional groups. We have confirmed that hADSC-Exos decrease glycogen synthase kinase-3β expression through the Wnt/β-catenin pathway, leading to increased proliferation and migration of dermal papillary cell. Excessive dihydrotestosterone can cause hair loss by shortening the hair growth phase, but hADSC-Exo treatment can reverse this effect. This study suggests that hADSC-Exo plays a role in hair regeneration through cell-to-cell communication via the Wnt/β-catenin pathway.
Citation: Fu Y, Han YT, Xie JL, Liu RQ, Zhao B, Zhang XL, Zhang J, Zhang J. Mesenchymal stem cell exosomes enhance the development of hair follicle to ameliorate androgenetic alopecia. World J Stem Cells 2025; 17(3): 102088
Hair loss causes a greater financial burden[1]. The two basic categories of hair loss are scarring and nonscarring alopecia and the latter is more common. Nonscarring alopecia can be categorized into androgenetic alopecia (AGA) and non-AGA, such as alopecia areata and telogen effluvium[2]. AGA is the most common form of nonscarring alopecia, affecting up to 50% of women and 80% of men, and the prevalence of AGA increases with age[3]. It is estimated that 0.2%-2% of the world’s population suffers from AGA, which affects 500000 men and 300000 women in the United States[4]. The causes of AGA are multifactorial, with genetic factors and androgens playing an important role in the development and progression of the disease. A recent United Kingdom biobank study showed a genealogical heritability of 0.62 and a single nucleotide polymorphism heritability of 0.39. A positive family history of AGA in Asian populations accounts for about 50% of cases[5]. AGA develops in 32.1%-63.3% of testosterone users[4]. By simple diffusion, testosterone enters the cell and is converted to dihydrotestosterone (DHT) by 5-a-reductase in the cytoplasm. When DHT binds to androgen receptors (ARs), it exerts transcriptional activity and contributes to the progression of AGA[6]. The dermal papillary cells (DPCs) of AGA patients have high levels of ARs, which makes them more sensitive to androgens[7]. Synthetic drugs, such as finasteride, used to treat hair loss, cause many side effects. Hence, it requires the development of new and efficient drugs to promote hair follicle development in AGA.
Mesenchymal stem cells (MSCs) and their secretomes show promise in promoting hair follicle regeneration. MSCs can be derived from a wide range of sources, such as umbilical cord, adipose tissue, amniotic fluid and bone marrow, which are named umbilical cord-derived MSCs (UCMSCs), adipose-derived MSCs (ADSCs) and bone marrow-derived MSCs, respectively[8,9]. Exosomes, 30-150 nm microvesicles, are actively released by viable cells and are composed of diverse proteins, encompassing signal proteins, noncoding RNAs and growth factors[10]. MSCs exert their therapeutic effect mainly through paracrine mechanisms, such as releasing exosomes[11]. Compared to MSCs, the exosomes derived from MSCs are cell-free and could be a promising therapy for AGA, with no major adverse effects[12]. However, various issues have restricted their clinical application.
MSCs exhibit heterogeneity at various levels, encompassing differences originating from subpopulations, donors, and different culture media[13,14]. With advances in sequencing techniques, liquid chromatography-mass spectrometry (LC-MS) proteome facilitates finding heterogeneity of MSCs. In previous studies, we obtained human adipose tissue from which human ADSCs (hADSCs) were isolated[15,16]. In this study, we obtained human UCMSCs (UCMSCs). We analyzed the protein composition of hADSC-exosomes (hADSC-Exos) and hUCMSC-Exos by LC-MS proteome analysis, so that the heterogeneity of exosomes with different cell origins was revealed. hADSC-Exos were also tested from the same donor source, cultured through three different culture systems, and selected proteins that were common to each culture system. In hADSC-Exos, 232 proteins were stably expressed and categorized into 10 functional groups. Additionally, as the DPCs are unique mesenchymal cells, they regulate hair follicle stem cells (HFSCs) during hair follicle regeneration and development[17]. Researchers have shown that exosomes derived from DPCs stimulate hair follicle development; therefore, we examined if hADSC-Exos improved DPC proliferation in AGA. There was a significant increase in Wnt3a in DPC cocultured with hADSC-Exos[18]. hADSC-Exo-derived cell division cycle protein 42 (CDC42) promoted DPC proliferation via the Wnt/β-catenin pathway. These findings provide novel insights into improving hair development in AGA.
MATERIALS AND METHODS
Cell culture
After obtaining high purity hADSCs and hUCMSCs, both cells were cultured in minimal essential medium MEMα with no Phenol-Red (Gibco, NY, United States). + 10% fetal bovine serum (FBS; ScienCell, CA, United States) and incubated at 37 °C with 5% CO2 for basal cell growth and proliferation. In subsequent analysis, hADSCs were cultured in three different culture systems, including basal culture medium (MEMα + 10% FBS), serum-free medium with phenol red (Cellartis® MSC Xeno-Free Culture Medium; Takara, Japan), and serum-free medium without phenol red (StemPro™ MSC SFM; Gibco, NY, United States) to explore the effect of different culture media on cellular exocytosis[19-21]. We purchased human hair DPCs (HHDPCs; ScienCell, CA, United States), which were obtained from a 58-year-old man. HHDPCs were cultured in MSC medium (ScienCell, CA, United States), containing 5% FBS, 1% MSC growth supplement (ScienCell, CA, United States) and 1% penicillin/streptomycin (ScienCell, CA, United States).
Exosome isolation
As described previously, the supernatant of hADSC/hUCMSC was obtained and subjected to centrifugation for eliminating suspended cells and cellular debris[22]. A 0.22-μm filter was used to filter the supernatant. Subsequently, the filtered supernatants were centrifuged using a Beckman Coulter ultra-high-speed centrifuge (Optima XPN-100 Ultracentrifuge; Beckman Coulter, Australia). Following centrifugation, Dulbecco phosphate buffer saline was introduced to cleanse the exosomes. Monitoring exosome uptake involves staining the exosomal membranes using fluorescent lipid membrane dye PKH26[23]. The hADSC-exos were processed using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, St. Louis, MO, United States). Finally, a microscope (Nikon, Japan) was used to observe the PKH26-hADSC-exos location.
Transmission electron microscopy
Refer to the methods, a copper mesh was used for aspiration of exosomes obtained through ultracentrifuge[24]. Following about 1 minute of operation, the surplus liquid was removed by filter paper positioned at the periphery of the mesh. Subsequently, phosphotungstic acid was applied to the copper mesh, and after 30 seconds, the excess liquid was eliminated using filter paper at the edge of the mesh. The sample was subjected to baking for 1 minute, and electron micrograph images were captured, using an 80-kV transmission electron microscope (Hitachi H-7650, Japan). The exosomes were observed as cup-shaped membrane vesicles ranging in size from 30 to 150 nm.
Nanoparticle tracking analysis
After washing the cells with deionized water, they were calibrated with polystyrene microspheres (110 nm). This was followed by washing and dilution with phosphate buffered saline. ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and NTA 3.0 software were used for nanoparticle tracking analysis (NTA)[25].
Western blotting
Total protein was extracted from the exosome samples using RIPA buffer, and the concentration was measured using the BCA protein assay. Western blotting with anti-CD9 and anti-CD81 was used to quantify total exosome proteins. Using rabbit anti-CD9 antibody (ab92726, 1:1000; Abcam, Cambridge, United Kingdom), rabbit anti-CD63 antibody (ab134045, 1:1000; Abcam, Cambridge, United Kingdom) for primary antibodies. Scanned images were acquired by Odyssey Infrared Imaging System (Licor, Lincoln, NE, United States).
Exosomal LC-MS proteome analysis
To commence the analysis, we introduced 30 μL of sample buffer (composed of acetonitrile, water, and formic acid in a ratio of 2:98:0.1) and agitated the mixture until the dried sample was fully dissolved. We subjected the sample to MS using an Easy-nLC 1000 system (Thermo Fisher Scientific, Bremen, Germany) equipped with a C18 reversed-phase column (PepMap100, C18 NanoViper, Thermofisher Dionex, CA, United States). The procedure involved a gradient from 2% to 40% of mobile phase B over a duration of 103 minutes. MS was conducted using a Q Exactive plus system (Thermo Scientific, CA, United States) featuring a nanoliter spray ESI ion source operating at a spray voltage of 1.6 kV. Three independent replicates from exosomes derived from control culture medium (FBS), phenol red culture medium (Takara, Japan), and phenol red free culture medium (Gibco, NY, United States) were analyzed for proteomics with Q Exactive plus (Thermo Fisher Scientific, Bremen, Germany). The different proteins in hADSC-Exo and hUCSMSC-Exo were listed in Supplementary Table 1.
Gene Ontology (GO) (https://geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://genome.jp/kegg) were used for selected proteins pathway analysis. The bubble diagram was drawn according to the R language ggplot2 package. The differential proteins screened were imported into STRING (https://string-db.org/). The STRING protein query database was used to build a protein-protein functional interaction network in Cytoscape cluster plugin. The clusters within the network were identified using the MCODE clustering algorithm.
Mice
Male 6-week-old C57BL/6 mice weighing 18-22 g were used. The mice were divided into the control, negative control, positive control and experimental groups (n = 6 per group). To visualize the initial follicle morphogenesis in the skin, all mice were shaved of their back hair, and 2-3 cm were removed. There was a pink coloration in the depilated areas of all the mice treated with depilatory cream, which suggests the dorsal skin was induced into anagen[26]. The control mice were only depilated for observation of normal hair follicle recovery after hair loss. For the negative control, positive control and experimental mice, all mice were established as AGA models. Testosterone propionate (TP, 5 mg/kg) was subcutaneously injected into the back once daily for 4 weeks[27,28]. To evaluate the effect of TP on hair follicle growth and development, we left the negative control group untouched. The first day of injection was marked as day 1. The positive control group was treated with minoxidil, an Food and Drug Administration (FDA)-approved drug for AGA[29]. The experimental group was treated with hADSC-Exos. Minoxidil and hADSC-Exos were started simultaneously on day 1 of TP injection, and ended on day 21. Mice were anesthetized by inhalation of 2% isoflurane before treatment. Microneedle (Mefonol disposable skin prick needle, Suzhou, China) treatment was selected. The specification was 540 rolling needles, 0.22-0.5 mm. To absorb minoxidil, the microneedle treatment head was gently rolled back and forth against the skin. The positive control group received 0.2 mL 5% minoxidil tincture daily, and the hADSC-Exo group was injected daily with a roller needle, as described previously[30,31]. A previous study demonstrated the optimal concentration of ADSC-Exos that promoted DPC proliferation[32]. We selected 100 μg/mL ADSC-Exos per mouse in the assay. The hair growth was photographed in all mice on day 21.
Macroscopic measurement for hair growth
Referred to the previous research, we recorded the time when the skin color changed from pink to black, and we rated and photographed the result on days 7, 14, and 21[33]. The method has been optimized as described by Kwon et al[34]. A score of 0 indicated that there was no hair growth in the depilated area and the epidermis was flesh-colored; 1 indicated that the epidermis of the depilated area was gray; 2 indicated that the epidermis of the depilated area was black; and 3 indicated that there was hair growth in the depilated area and the epidermis was black. Referred to the previous research, hair follicles that formed in the new tissue at day 21 were counted on three randomly selected hematoxylin and eosin-stained sections per animal[35].
Cell proliferation
Enhanced cell counting kit-8 (CCK8, Sangon Biotech, China) was used to assess proliferation of DPCs. DPCs were seeded in 96-well plates that were incubated with CCK8 reagents (100 μL/well) for 1 hour and detected at 0, 24, and 48 hours. The results were quantitated using a 450 nm microplate reader.
Cell migration
Transwell assay was used to assess migration of DPCs by Corning TransWell Chamber (Corning, NY, United States). After trypsinization, counting and incubation in 100 μL medium without FBS, DPCs were collected in a 24-well plate (2 × 105 cells/well). In the lower chamber, 800 μL medium supplemented with 30% FBS was added. The migratory capacity of cells was assessed by fixing and staining with 4% formaldehyde and crystal violet solution (Biyuntian, China).
Quantitative polymerase chain reaction
Total RNA isolation was performed using QIAGEN miRNeasy Mini (Valencia, CA, United States). Reverse transcription was performed using a PrimeScript RT Master Mix (Takara, Shiga, Japan). Quantitative polymerase chain reaction was performed using an ABI7500 instrument (Oyster Bay, NY, United States).
RESULTS
Isolation and validation of hADSC-Exos and hUCMSC-Exos
To clarify the heterogeneity between MSCs from different cell sources, we cultured hADSCs and hUCMSCs. Microscopic images revealed that the hADSCs had a typical long spindle-shaped morphology, consistent with the morphological characterization of hUCMSCs (Figure 1A). hADSCs and hUCMSCs showed high levels of MSC marker expression, such as CD73, CD90, CD105, CD45, and HLA-DR (Figure 1B). hADSC-Exos and hUCMSC-Exos were extracted using ultra-high-speed centrifugation (Figure 1C). Transmission electron microscopy revealed a bowl-shaped structure of the hADSC-Exos and hUCMSC-Exos (Figure 1D). NTA demonstrated that the diameter of the hADSC-Exos was about 100 nm, falling within the size range criteria for exosomes (30-150 nm). NTA revealed that the average diameter of hUCMSC-Exos was 139 nm (Figure 1E). HADSC-Exos expressed the exosome markers CD9, CD63, and CD81 (Figure 1F). These results indicated that our exosomes met the morphological characteristics of hADSC-Exos and hUCMSC-Exos.
Figure 1 Identification of exosome from human adipose-derived mesenchymal stem cell and human umbilical cord-derived mesenchymal stem cell.
A: Morphology of human adipose-derived mesenchymal stem cell (hADSC) and human umbilical cord-derived mesenchymal stem cell (hUCMSC); B: Analysis of surface markers on hADSCs and hUCMSCs showed high CD105, CD90, and CD73 expression, but negative HLA-DR and CD45 expression; C: Schematic presentation of exosome isolated from hADSC and hUCMSC by differential ultracentrifugation; D: By TEM, purified hADSC exosome (hADSC-Exo) and hUCMSC exosome (hUCMSC-Exo) exhibit cup-like morphologies; E: Nanoparticle analysis of hADSC-Exo and hUCMSC-Exo; F: hADSC-Exo and hUCMSC-Exo express CD63, CD9, CD81 and calnexin is not expressed. hADSC: Human adipose-derived mesenchymal stem cell; hUCMSC: Human umbilical cord-derived mesenchymal stem cell; hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; hUCMSC-Exo: Human umbilical cord-derived mesenchymal stem cell exosome.
LC-MS proteome analysis to identify the common function of hADSC-Exos and hUCMSC-Exos
LC-MS proteome analysis was performed to explore the similarities and differences between hADSC-Exos and hUCMSC-Exos. hADSC-Exos included 724 proteins, while hUCMSC-Exos includes 1246. Six hundred and fifty overlapped proteins were obtained by investigating the intersection of the hADSC-Exos and hUCMSC-Exos (Figure 2A). The 650 overlapped proteins were used for GO term enrichment and KEGG pathway enrichment analysis to explore the common function of hADSC-Exos and hUCMSC-Exos. The GO biological process analysis revealed that the shared proteins were significantly associated with signal transduction, cell adhesion and cell-cell adhesion (Figure 2B). The GO cellular component analysis suggested that the common proteins were significantly associated with extracellular exosomes, cytosol and cytoplasm (Figure 2C). The GO molecular function analysis revealed that the common proteins were significantly associated with protein binding, poly(A) RNA binding and ATP binding (Figure 2D). KEGG pathway analysis showed that regulation of actin cytoskeleton, focal adhesion and phosphatidylinositol 3-kinase/protein kinase B signaling pathway were the main pathways (Figure 2E). These results illustrate potentially shared functional commonalities between hADSC-Exos and hUCMSC-Exos.
Figure 2 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis based on liquid chromatography-mass spectrometry proteome analysis data of human adipose-derived mesenchymal stem cell exosome and human umbilical cord-derived mesenchymal stem cell exosome.
A: Venn map showing the intersection proteins of human adipose-derived mesenchymal stem cell exosome (hADSC-Exo) and human umbilical cord-derived mesenchymal stem cell exosome (hUCMSC-Exo); B-D: Gene Ontology analysis of hADSC-Exo and hUCMSC-Exo liquid chromatography-mass spectrometry common proteins. A chart indicates biological process (B), cellular components (C) and molecular function (D); E: Kyoto Encyclopedia of Genes and Genomes pathway of hADSC-Exo and hUCMSC-Exo common proteins. hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; hUCMSC-Exo: Human umbilical cord-derived mesenchymal stem cell exosome; LC-MS: Liquid chromatography-mass spectrometry; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; CC: Cellular component; MF: Molecular function.
LC-MS proteome analysis of heterogeneous characteristics in hADSC-Exos and hUCMSC-Exos
Despite the functional commonalities between hADSC-Exos and hUCMSC-Exos, their specific functional differences have yet to be elucidated. The GO biological process analysis revealed that the specific proteins in hADSC-Exos were significantly associated with nucleosome assembly, blood coagulation and cell adhesion, while the specific proteins in hUCMSC-Exos were significantly associated with cell adhesion, cell-cell adhesion and proteolysis (Figure 3A and B). The GO cellular component analysis suggested that the specific proteins in hADSC-Exos and hUCMSC-Exos were related to extracellular exosomes and cytosol (Figure 3C and D). The GO molecular function analysis revealed that the specific proteins in hADSC-Exos were significantly associated with calcium ion binding, protein heterodimerization activity and nucleosomal DNA binding, while the specific proteins in hUCMSC-Exos were related to protein binding, RNA binding and ATP poly(A) binding (Figure 3E and F). The specific KEGG pathway in hADSC-Exos were metabolic pathways, systemic lupus erythematosus and alcoholism (Figure 3G). The specific KEGG pathway in hUCMSC-Exos were phosphatidylinositol 3-kinase/protein kinase B signaling, endocytosis and biosynthesis of antibiotics (Figure 3H). The LC-MS proteome analysis results were validated through quantitative real-time polymerase chain reaction (Figure 3I and J). hADSC-Exos mainly participated in blood coagulating, skin wound repair and inflammatory reaction. These findings suggest the beneficial role of hADSC-Exos in promoting skin repair. We then investigated whether hADSC-Exos affected the development of skin or hair follicles.
Figure 3 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis based on heterogeneous characteristics of human adipose-derived mesenchymal stem cell exosomes and human umbilical cord-derived mesenchymal stem cell exosome.
A and B: Heterogeneous characteristics of human adipose-derived mesenchymal stem cell exosome (hADSC-Exo) (A) and human umbilical cord-derived mesenchymal stem cell exosome (hUCMSC-Exo) (B) function analyzed by biological process analysis; C and D: Heterogeneous characteristics of hADSC-Exo (C) and hUCMSC-Exo (D) function analyzed by cellular components analysis; E and F: Heterogeneous characteristics of hADSC-Exo (E) and hUCMSC-Exo (F) function analyzed by molecular function analysis; G and H: Heterogeneous characteristics of hADSC-Exo (G) and hUCMSC-Exo (H) function analyzed by Kyoto Encyclopedia of Genes and Genomes pathway analysis; I and J: The mRNA expression of COL7A1 (hADSC-Exo, I) and PSMD2 (hUCMSC-Exo, J) was detected by reverse transcription-polymerase chain reaction (data were presented as mean ± SD. bP < 0.01, n = 6, unpaired t-test. hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; hUCMSC-Exo: Human umbilical cord-derived mesenchymal stem cell exosome; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; CC: Cellular component; MF: Molecular function.
hADSC-Exos improved hair development in AGA mouse model
There are three phases of the hair follicle, anagen (active growth stage), catagen (regression of the hair follicle) and telogen (resting stage). There is a shorter anagen stage of hair follicles in the AGA patient’s area of baldness, but a longer resting stage. Hair eventually fails to grow from the surface of the skin when the anagen period is too short[36]. The next step was to examine whether hADSC-Exos promoted hair growth in the AGA mouse model. Subcutaneous TP injections were administered daily in the dorsally depilated area to the androgen, positive control, and hADSC-Exos groups (Figure 4A). Immunofluorescence revealed that PKH26-labeled hADSC-Exos had the capability to enter the skin. Keratin K14, which highlights the epidermis and hair follicles, was the green counterstain (Figure 4B). To evaluate hair growth score and the number of follicles, skin samples were collected 21 days after depilation. Adult C57BL/6 mice with dorsal hair depilated had pink skin in the telogen phase after their dorsal hair was removed (Figure 4C). After 7 days, hair follicles re-entered anagen, resulting in dark grey skin[37]. The TP-treated mice still had pink backs, indicating that the hair follicles were still resting. Except for the TP-treated group, the hair of all mice at 14 days was mostly intact, suggesting that exosomes resisted androgens and promoted anagen hair growth. Hematoxylin and eosin staining of the hair shafts and photographs revealed the number of hair follicles (Figure 4D). The TP-treated hADSC-Exo group had significantly more hair development than the TP-treated group at 14 days (Figure 4E). The number of anagen hair follicles was similar between the positive control and hADSC-Exo groups after 21 days, and it significantly increased compared with the TP-treated group (Figure 4F). These findings suggested that hADSC-Exos played a beneficial role in promoting hair follicle development in an AGA mouse model in vivo.
Figure 4 Human adipose-derived mesenchymal stem cell exosomes improve the hair regeneration in androgenetic alopecia mice model.
A: Schematic flowchart of the experiment of testosterone propionate-treated and shaved in androgenetic alopecia mouse model; B: PKH26-labeled human adipose-derived mesenchymal stem cell exosome (red) retention in the site of hair follicle; C: Hair growth of C57BL/6 mice in each group; D: hematoxylin and eosin staining of mouse skin histopathological sections in each group; E: Hair growth score in each group; F: The number of follicles in each group. Differences among four groups were assessed by Tukey’s multiple comparison test and one-way ANOVA; error bars represent SEM. bP < 0.01, compared with testosterone propionate-treated group. Six mice were randomly selected from each group for histological examination. TP: Testosterone propionate; hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome.
Effective components of hADSC-Exos can modulate cell cycle
We performed LC-MS proteomic analysis on hADSC-Exos isolated from different ADSC culture media. A Venn diagram was drawn to find the common proteins of FBS-cultured hADSC-Exos, phenol red medium-cultured hADSC-Exos, and phenol red free medium-cultured hADSC-Exos. FBS-cultured hADSC-Exos included 621 proteins, phenol red medium-cultured hADSC-Exos 1026 proteins, and 302 proteins were found in phenol red free medium-cultured hADSC-Exos (Figure 5A). A total of 232 proteins were shared among the three different culture media, which were probably the main proteins in hADSC-Exos. The co-expressed proteins were analyzed by GO and KEGG pathway using the David database (https://david.ncifcrf.gov/) to further understand the biological functions of exosomes. The molecular functions of these co-detected proteins in hADSC-Exos were mainly protein heterodimerization activity, cell adhesion molecule binding, nucleosomal DNA binding, and GTP binding (Figure 5B). The cellular components involved in the exosomes were mainly the nucleosome, DNA packaging complex and protein-DNA complex (Figure 5C). The biological processes involved in the exosomes were mainly chromatin silencing, nucleosome assembly, and negative regulation of epigenetic gene expression (Figure 5D). KEGG pathway enrichment analysis was performed on shared proteins, and the 15 pathways with the smallest P value were selected for bubble mapping, which indicated that the signaling pathways involved in exosomes were systemic lupus erythematosus, alcoholism and viral carcinogenesis. The 232 active proteins mentioned above were subjected to protein interactions network analysis using the String website, followed by annotation analysis of the proteins with GeneCards (https://www.genecards.org), and Ctyoscape software for analysis and mapping. These 232 active proteins were categorized into 10 groups according to their functions: Metabolism, complement and coagulation cascades, extracellular matrix (ECM), cell cycle, protein transport from cytoplasm to nucleus, post-translational modifications, nucleosome assembly, proteasome and exosome biogenesis (Figure 5E). We focused on the cell cycle group. Some cell cycle regulatory genes promoted cell growth, such as CDC42, RhoA, and Stratifin (SFN) (Figure 5F). The Rho GTPase family member CDC42 is essential for progression through G1[38]. Transient interactions between CDC42 and its downstream effector proteins are induced by various stimuli, and its deficiency might be associated with skin barrier damage or dysfunction[39,40]. Keratinocyte-specific deletion of RhoA promotes increased tumor growth, less differentiation and invasiveness in a mouse model of skin cancer[41]. SFN, also called 14-3-3σ protein, has been shown to be effective in treating UVB-induced skin disease. SFN regulates cellular activities such as cell cycle, cell growth, cell survival, and gene transcription[42]. These results suggest that hADSC-Exos play an essential role in maintaining the cell cycle.
Figure 5 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis based on liquid chromatography-mass spectrometry proteome analysis data of human adipose-derived mesenchymal stem cell exosomes in different culture medium.
A: Venn map showing the intersection of human adipose-derived mesenchymal stem cell exosomes (hADSC-Exo) proteins in different culture systems: Control medium (fetal bovine serum), phenol red free culture medium (Gibco, NY, United States), and phenol red culture medium (Takara, Japan); B-D: Gene Ontology analysis of hADSC-Exo liquid chromatography-mass spectrometry proteome analysis data. A chart indicates molecular function (B), cellular components (C) and biological process (D); E: Kyoto Encyclopedia of Genes and Genomes pathway of hADSC-Exo proteins; F: STRING network analysis of the intersection of hADSC-Exo proteins. hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; LC-MS: Liquid chromatography-mass spectrometry; FBS: Fetal bovine serum.
hADSC-Exos improved hair development in AGA cell models
Increasing evidence indicates that DPCs stimulate hair regeneration. We investigated whether hADSC-Exos promoted hair regeneration by affecting HHDPCs. We analyzed HHDPCs in conventional culture at passage three. HHDPCs displayed long spindle‐like shapes, formed colonies and reached confluency (Figure 6A). Referred to the previous research, we confirmed the transfection operations and reagent dosage[43]. The control group was untreated HHDPCs, and 100 nmol/L DHT was used in the AGA cell model group. To investigate the effect of hADSC-Exos on HHDPC proliferation, the CCK8 assay was performed. Growth curve assay based on CCK8 analysis was tested at 24 and 48 hours after hADSC-Exo treatment (Figure 6B and C). hADSC-Exos increased HHDPC proliferation over 48 hours (Figure 6B). Proliferation of human DPCs was inhibited by DHT as compared with the control group at 48 hours (Figure 6C). hADSC-Exos rescued the proliferation of DHT-treated HHDPCs (Figure 6C). Migration of HHDPCs transfected with hADSC-Exos was assessed by scratch wound assay and Transwell assay. Likewise, the migratory capacity was restored by overexpressing hADSC-Exos in DHT-treated DPCs in vitro (Figure 6D-G). These findings suggested that hADSC-Exos played a beneficial role in promoting hair follicle development in an AGA cell model in vitro.
Figure 6 Human adipose-derived mesenchymal stem cell exosome improves the hair regeneration in androgenetic alopecia cell model.
A: Human hair dermal papillary cells were used in subsequent analyses from passage 3 (P3); B and C: Cell counting kit-8 (CCK8) assay. CCK8 assay was carried out to measure the cell growth in 48 hours (B). Cell survival was determined by the CCK8 assay at 48 hours (C); D and E: The wound healing assay was used to assess migration in cells (D). Statistical analysis of the results of wound healing assay in each group (E); F and G: The migratory properties of human hair dermal papillary cells were analyzed using the Transwell migration assay with Transwell filter chambers (F). Statistical analysis of the results of Transwell migration assay in each group (G). bP < 0.01, cP < 0.001, dP < 0.0001. HHDPC: Human hair dermal papillary cell; DHT: Dihydrotestosterone; hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome.
hADSC-Exo regulated DPC proliferation via the CDC42/Wnt/β-catenin pathway in AGA
The key genes in AGA are listed in Supplementary Table 2. By overlapping the cell cycle genes in hADSC-Exos with AGA-related genes, we identified six candidate genes (Figure 7A). CDC42 has been reported as the protein that promotes ADSC-derived insulin-producing cell proliferation via Wnt/β-catenin signaling[44]. In addition, the cell cycle related Wnt pathway in involved in 5 biological pathways for CDC42 gene (Figure 7B). DHT signaling activates glycogen synthase kinase (GSK)-3β present in DPCs, whereas receptor AR signaling leads to phosphorylation of β-catenin to reduce its level, antagonizing the classical Wnt signaling pathway[45]. Therefore, we used Hair-GEL single cell databases and analyzed the expression of CDC42 and GSK-3β (Figure 7C and D). The expression of CDC42 in DPCs suggested the role of CDC42 in the DPC cell cycle, and GSK-3β was also expressed in DPCs. Under the effect of hADSC-Exos, expression level of Wnt3a and β-catenin in DPCs was significantly increased compared with the control group. Under the effect of DHT, expression of Wnt3a and β-catenin decreased compared with that of the control group. The expression of Wnt3a and β-catenin in the hADSC-Exo- and DHT-treated groups increased compared with that in the DHT-treated alone group (Figure 7E-H). By activating the Wnt/β-catenin signaling pathway through CDC42, hADSC-Exos can potentially suppress GSK-3β expression, counteracting the inhibitory impact of DHT, stimulating cell proliferation, and enhancing hair growth (Figure 8).
Figure 7 Human adipose-derived mesenchymal stem cell exosomes regulate dermal papillary cell proliferation via cell division cycle protein 42/Wnt/β-catenin pathway in androgenetic alopecia.
A: Venn diagram demonstrating the intersections of genes from the androgenetic alopecia gene set and human adipose-derived mesenchymal stem cell exosomes cell cycle gene; B: Five-sino biological pathway enrichment diagram; C: Using single-cell mRNA-sequencing data from the Hair-GEL database, cell types expressing cell division cycle protein 42 (http://hair-gel.net/) were identified; D: Using single-cell mRNA-sequencing data from the Hair-GEL database, cell types expressing glycogen synthase kinase-3β (Gsk3β) (http://hair-gel.net/) were found; E: Representative western blotting image of GSK-3β after human adipose-derived mesenchymal stem cell exosomes was overexpressed in human hair dermal papillary cells; F-H: Statistical analysis of the western blotting in each group. Wnt3a (F), GSK-3β (G) and β-catenin (H). Differences among five groups were assessed by Tukey’s multiple comparison test and one-way ANOVA, and error bars represent SEM. bP < 0.01, cP < 0.001, dP < 0.0001, Compared with control group. hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; AGA: Androgenetic alopecia; Cdc42: Cell division cycle protein 42; Gsk3b: Glycogen synthase kinase-3β; DHT: Dihydrotestosterone.
Figure 8 Human adipose-derived mesenchymal stem cell exosome regulate dermal papillary cells proliferation via Wnt/β-catenin/glycogen synthase kinase-3β in hair follicle development.
DPC: Dermal papillary cell; hADSC-Exo: Human adipose-derived mesenchymal stem cell exosome; CDC42: Cell division cycle protein 42; GSK3β: Glycogen synthase kinase-3β; DHT: Dihydrotestosterone.
DISCUSSION
We explored the effect of hADSC-Exos on hair follicle development in AGA. It is believed that AGA is caused by both hereditary and androgenic factors. TP is one of the common androgens that induce AGA, which is a slow release formulation of testosterone. Testosterone is metabolized to DHT by steroid 5α-reductases[46]. DHT has a 10-fold higher potency than testosterone as the primary AR ligand[47]. DHT inhibits proliferation of HaCaT keratinocytes in coculture with DPCs obtained from AGA patients, by suppressing the Wnt signaling pathway[48]. The Wnt/β-catenin signaling pathway plays an important role in cell proliferation, differentiation, apoptosis and stem cell renewal. Researchers have found that Wnt signaling can induce stem cells to differentiate into sebaceous glands and hair follicles[49]. In summary, compared with FDA-approved minoxidil, our results have proved that hADSC-Exos have an ameliorative effect. We found that hADSC-Exos inhibited the expression of GSK-3β by activating the Wnt/β-catenin signaling pathway through CDC42.
At present, the only therapeutic agents for AGA approved by the FDA in the United States are finasteride and minoxidil. However, finasteride is only effective in male subjects[50]. Minoxidil cannot prevent hair loss but may accelerate hair growth, but only 13%-40% of female subjects with AGA responded to minoxidil treatment[51]. The limitations of these drugs make it necessary to develop a more effective alternative. AGA treatments have significantly evolved over the past few decades, incorporating a range of techniques and technologies, such as platelet-rich plasma (PRP), microneedling, and low-level laser therapy and stem cell therapy[52]. PRP therapy utilizes autologous blood components enriched with growth factors, promoting hair regrowth by enhancing follicular health and stimulating hair follicle anagen phase. Clinical studies have demonstrated the effectiveness of PRP in enhancing hair density and thickness in both AGA and female alopecia[53,54]. Microneedling involves creating microinjuries in the scalp to stimulate hair follicles and improve the absorption of topical treatments. This technique improves scalp health and can be a valuable adjunct to other hair restoration methods[55]. Low-level laser therapy stimulates hair follicles with low-intensity lasers. It has been shown to increase hair density and promote regrowth through enhanced cellular metabolism and reduced inflammation[56]. Stem cell therapy may improve hair regrowth by reversing the pathological mechanisms or regulating cellular quiescence. HFSCs and MSCs are the most widely used stem cells for the study of AGA. HFSCs can effectively induce hair regrowth by reactivating the hair growth cycle and enhancing follicle development[57]. MSCs are another promising cell-based therapy for hair restoration, such as ADSCs and UCMSCs. Their regenerative potential stems from their ability to differentiate into various cell types and release growth factors that promote hair follicle development[58]. Despite the significant impact of MSCs on hair loss or thinning, there are some clinical limitations[59-61]. Compared to MSC therapy, exosome-based cell-free therapy is more stable in clinical application[61].
The importance of homogeneous MSC-Exos should be emphasized, which is one of the prerequisite steps in further clinical treatment of AGA. MSCs and their exosome have their own advantages and disadvantages in terms of isolation, differentiation capacity and cell count, as well as possible side effects[58]. This heterogeneity underscores the importance of establishing standardized quality control protocols for the clinical application of MSC-Exos. Incorporating proteomics as a quality control step may prove to be valuable in this regard. Here, we used a standardized method to obtain hADSC-Exos and hUCMSC-Exos with safety and stability, and selected hADSC-Exos by LC-MS proteomic analysis. The process of obtaining ADSCs involves liposuction, which is a less invasive procedure. This makes the procedure more acceptable to patients and reduces the risk of complications associated with the cell harvesting process. In addition, since ADSCs are derived from the patient’s own adipose tissue, the risk of immunological rejection is minimal[62]. Our findings indicate that hADSC-Exos are more effective in promoting hair follicle development compared to hUCMSC-Exos. Several proteins associated with angiogenesis, inflammation regulation, and ECM remodeling may also play an important role in hADSC-Exos, as there may be a potential impact of these proteins on HFSC activation, as well as on transition of hair follicles from the resting to the anagen phase. For instance, the aging of hair follicles is characterized by a reduction in the expression of cell adhesion and ECM genes in HFSCs, which are controlled by nuclear factor of activated T cells cytoplasmic 1 and forkhead box protein C1[63]. ADSCs have been shown to promote vascularization and regulate immunological responses through paracrine signaling, which could be beneficial in managing inflammation during the anagen phase[64]. Overall, hADSC-Exos can provide insights into potential therapeutic strategies for enhancing hair growth and regeneration.
The clinical applicability of hADSC-Exo therapy holds great promise, but it requires careful consideration of administration methods, dosage optimization, long-term effects, and safety[61]. Administration of hADSC-Exos can be performed through various routes, including topical, intravenous and subcutaneous methods. For instance, topical application may be more suitable for localized conditions such as hair growth and wound healing. Dose optimization is essential to maximize therapeutic benefits while minimizing potential side effects. This requires extensive preclinical and clinical studies to establish dose-response relationships and identify the most effective and safe dosage regimens[65]. The long-term effects of hADSC-Exo therapy are still under investigation. However, studies have shown that exosomes generally exhibit low immunogenicity, which is promising for their long-term use[66]. Lastly, safety considerations are paramount in the clinical translation of hADSC-Exo therapy. It is crucial to conduct comprehensive safety evaluations, including assessments of potential tumorigenicity, immunogenicity, and other adverse effects.
CONCLUSION
In summary, there are many advantages to ADSCs and their exosomes, such as their robust regenerative potential, minimal invasive harvesting, sustained efficacy, and synergistic potential. They can be used in combination with other hair regrowth treatments, such as PRP and microneedling, to enhance overall effectiveness. Here, we used needle roller administration. In the future, the effects of different modes of hADSC-Exo administration on hair follicle development should continue to be explored. Clinical trials must adhere to standardization of isolation techniques, culture media used, dose and other critical information.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Cell and tissue engineering
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade A, Grade B, Grade B, Grade B, Grade C
Novelty: Grade B, Grade B, Grade B, Grade C
Creativity or Innovation: Grade B, Grade B, Grade C, Grade C
Scientific Significance: Grade B, Grade B, Grade C, Grade C
P-Reviewer: Duan Y; Jafar Sameri M; Kim DK; Li SC S-Editor: Wang JJ L-Editor: A P-Editor: Zhang XD
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