DNA methylation of microRNA-365-1 induces apoptosis of hair follicle stem cells by targeting DAP3.

Background: DNA methylation is a crucial epigenetic alteration involved in diverse biological processes and diseases. Nevertheless, the precise role of DNA methylation in chemotherapeutic drug-induced alopecia remains unclear. This study examined the role and novel processes of DNA methylation in regulating of chemotherapeutic drug-induced alopecia. Methods: A mouse model of cyclophosphamide (CTX)-induced alopecia was established. Hematoxylin-eosin staining and immunohistochemical staining for the Ki67 proportion and a mitochondrial membrane potential assay (JC-1) were performed to assess the structural integrity and proliferative efficiency of the hair follicle stem cells (HFSCs). Immunofluorescence staining and real-time fluorescence quantitative PCR (RT-qPCR) were performed to determine the expression levels of key HFSC markers, namely Lgr5, CD49f, Sox9, CD200, and FZD10. Differential DNA methylation levels between the normal and CTX-induced model groups were determined through simple methylation sequencing and analyzed using bioinformatics tools. The expression levels of miR-365-1, apoptosis markers, and DAP3 were detected through RT-qPCR and western blotting. In parallel, primary mouse HFSCs were extracted and used as a cell model, which was constructed using 4-hydroperoxycyclophosphamide. The luciferase reporter gene assay was conducted to confirm miR-365-1 binding to DAP3. To measure the expression of relevant indicators, superoxide dismutase (SOD) and malondialdehyde (MDA) kits were used. Methylation-specific PCR (MS-PCR) was performed to determine DNA methylation levels. The regulatory relationship within HFSCs was confirmed through plasmid overexpression of miR-365-1 and DAP3. Result: In the alopecia areata model, a substantial number of apoptotic cells were observed within the hair follicles on the mouse backs. Immunofluorescence staining revealed that the expression of HFSC markers significantly reduced in the CTX group. Both RT-qPCR and western blotting demonstrated a noteworthy difference in DNA methyltransferase expression. Simple methylation sequencing unveiled that DNA methylation substantially increased within the dorsal skin of the CTX group. Subsequent screening identified miR-365-1 as the most differentially expressed miRNA. miR-365-1 was predicted and confirmed to bind to the target gene DAP3. In the CTX group, SOD and ATP expression markedly reduced, whereas MDA levels were significantly elevated. Cellular investigations revealed 4-HC-induced cell cycle arrest and decreased expression of HFSC markers. MS-PCR indicated hypermethylation modification of miR-365-1 in the 4-HC-induced HFSCs. The luciferase reporter gene experiment confirmed the binding of miR-365-1 to the DAP3 promoter region. miR-365-1 overexpression dramatically reduced apoptotic protein expression in the HFSCs. However, this effect was slightly reversed after DAP3 overexpression in lentivirus. Conclusion: This study explored the occurrence of miR-365-1 DNA methylation in chemotherapeutic drug-induced alopecia. The results unveiled that miR-365-1 reduces cell apoptosis by targeting DAP3 in HFSCs, thereby revealing the role of DNA methylation of the miR-365-1 promoter in chemotherapeutic drug-induced alopecia.

Salvianolic Acid B Reduces Oxidative Stress to Promote Hair-Growth in Mice, Human Hair Follicles and Dermal Papilla Cells.

Background: Existing research links oxidative stress and inflammation to hair loss. Salvianolic acid B (SAB) is known for its anti-oxidative, anti-inflammatory, and other beneficial pharmacological properties. Objective: To assess the efficacy of SAB in modulating hair growth. Methods: In vivo experiments were conducted using C57BL/6 mice to evaluate the effects of SAB on hair and skin parameters. The study involved ex vivo analysis of human hair follicles (HFs) for hair shaft length and hair growth cycle assessment. In vitro, human dermal papilla cells (hDPCs) were cultured with SAB, and their proliferation, protection against H2O2-induced oxidative damage, and gene/protein expression alterations were examined using various analytical techniques, including Real-Time Cell Analysis (RTCA), DCFH-DA Assay, RNA-seq, and KEGG pathway analysis. Results: SAB treatment in mice significantly improved hair growth and vascularization by day 21. In human HFs, SAB extended hair shaft length and delayed the transition to the catagen phase. SAB-treated hDPCs showed a notable decrease in the expression of oxidation-antioxidation-related genes and proteins, including reduced phosphorylation levels of ERK and p38. Conclusion: The study indicates that SAB promotes hDPC proliferation and offers protection against oxidative stress, highlighting its potential as a therapeutic agent for enhancing hair growth and treating hair loss.

An optimized protocol for generating appendage-bearing skin organoids from human-induced pluripotent stem cells.

Organoid generation from pluripotent stem cells is a cutting-edge technique that has created new possibilities for modelling human organs in vitro, as well as opening avenues for regenerative medicine. Here, we present a protocol for generating skin organoids (SKOs) from human-induced pluripotent stem cells (hiPSCs) via direct embryoid body formation. This method provides a consistent start point for hiPSC differentiation, resulting in SKOs with complex skin architecture and appendages (e.g. hair follicles, sebaceous glands, etc.) across hiPSC lines from two different somatic sources.

Integrating Single-Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation.

The dermis and epidermis, crucial structural layers of the skin, encompass appendages, hair follicles (HFs), and intricate cellular heterogeneity. However, an integrated spatiotemporal transcriptomic atlas of embryonic skin has not yet been described and would be invaluable for studying skin-related diseases in humans. Here, single-cell and spatial transcriptomic analyses are performed on skin samples of normal and hairless fetal pigs across four developmental periods. The cross-species comparison of skin cells illustrated that the pig epidermis is more representative of the human epidermis than mice epidermis. Moreover, Phenome-wide association study analysis revealed that the conserved genes between pigs and humans are strongly associated with human skin-related diseases. In the epidermis, two lineage differentiation trajectories describe hair follicle (HF) morphogenesis and epidermal development. By comparing normal and hairless fetal pigs, it is found that the hair placode (Pc), the most characteristic initial structure in HFs, arises from progenitor-like OGN+/UCHL1+ cells. These progenitors appear earlier in development than the previously described early Pc cells and exhibit abnormal proliferation and migration during differentiation in hairless pigs. The study provides a valuable resource for in-depth insights into HF development, which may serve as a key reference atlas for studying human skin disease etiology using porcine models.

Pilose antler extract promotes hair growth in androgenic alopecia mice by promoting the initial anagen phase.

Androgenetic alopecia (AGA) is a prevalent disease in worldwide, local application or oral are often used to treat AGA, however, effective treatments for AGA are currently limited. In this work, we observed the promoting the initial anagen phase effect of pilose antler extract (PAE) on hair regeneration in AGA mice. We found that PAE accelerated hair growth and increased the degree of skin blackness by non-invasive in vivo methods including camera, optical coherence tomography and dermoscopy. Meanwhile, HE staining of sagittal and coronal skin sections revealed that PAE augmented the quantity and length of hair follicles, while also enhancing skin thickness and hair papilla diameter. Furthermore, PAE facilitated the shift of the growth cycle from the telogen to the anagen phase and expedited the proliferation of hair follicle stem cells and matrix cells in mice with AGA. This acceleration enabled the hair follicles to enter the growth phase at an earlier stage. PAE upregulated the expression of the sonic hedgehog (SHH), smoothened receptor, glioma-associated hemolog1 (GLI1), and downregulated the expression of bone morphogenetic protein 4 (BMP4), recombinant mothers against decapentaplegic homolog (Smad) 1 and 5 phosphorylation. This evidence suggests that PAE fosters hair growth and facilitates the transition of the growth cycle from the telogen to the anagen phase in AGA mice. This effect is achieved by enhancing the proliferation of follicle stem cells and matrix cells through the activation of the SHH/GLI pathway and suppression of the BMP/Smad pathway.

Single-cell transcriptome profiling reveals cell type-specific variation and development in HLA expression of human skin.

Skin, the largest organ of body, is a highly immunogenic tissue with a diverse collection of immune cells. Highly polymorphic human leukocyte antigen (HLA) molecules have a central role in coordinating immune responses as recognition molecules. Nevertheless, HLA gene expression patterns among diverse cell types within a specific organ, like the skin, have yet to be thoroughly investigated, with stromal cells attracting much less attention than immune cells. To illustrate HLA expression profiles across different cell types in the skin, we performed single-cell RNA sequencing (scRNA-seq) analyses on skin datasets, covering adult and fetal skin, and hair follicles as the skin appendages. We revealed the variation in HLA expression between different skin populations by examining normal adult skin datasets. Moreover, we evaluated the potential immunogenicity of multiple skin populations based on the expression of classical HLA class I genes, which were well represented in all cell types. Furthermore, we generated scRNA-seq data of developing skin from fetuses of 15 post conception weeks (PCW), 17 PCW, and 22 PCW, delineating the dynamic expression of HLA genes with cell type-dependent variation among various cell types during development. Notably, the pseudotime trajectory analysis unraveled the significant variance in HLA genes during the evolution of vascular endothelial cells. Moreover, we uncovered the immune-privileged properties of hair follicles at single-cell resolution. Our study presents a comprehensive single-cell transcriptomic landscape of HLA genes in the skin, which provides new insights into variation in HLA molecules and offers a clue for allogeneic skin transplantation.

Effect of N-acetylcysteine on hair follicle changes in mouse model of cyclophosphamide-induced alopecia: histological and biochemical study.

Chemotherapy-induced alopecia (CIA) represents one of the most severe side effects of chemotherapy, which forces some patients to reject cancer treatment. The exact pathophysiological mechanisms of CIA are not clearly understood, which makes it difficult to discover efficient preventive or therapeutic procedures for this adverse effect. N-acetylcysteine (NAC) has a strong antioxidant activity as it stimulates glutathione synthesis and acts as an oxygen radical scavenger. The current study tried to investigate the efficacy of NAC in preserving biochemical parameters and hair follicle structure against cyclophosphamide (CYP) administration. In total, 40 adult female C57BL/6 mice were induced to enter anagen by depilation (day 0) and divided into four groups: group I (control), group II (CYP) received a single dose of CYP [150 mg/kg body weight (B.W.)/intraperitoneal injection (IP)] at day 9, group III (CYP & NAC) received a single dose of CYP at day 9 as well as NAC (500 mg/kg B.W./day/IP) from day 6-16, and group IV (NAC) received NAC from day 6-16. CYP administration in group II induced an increase in malondialdehyde (MDA), decrease in superoxide dismutase (SOD), histological hair follicle dystrophy, disruption of follicular melanogenesis, overexpression of p53, and loss of ki67 immunoreactivity. NAC coadministration in group III reversed CYP-induced alterations in the biochemical parameters and preserved hair follicle structure, typical follicular melanin distribution as well as normal pattern of p53 and ki67 expression. These findings indicated that NAC could be used as an efficient and safe therapeutic option for hair loss induced by chemotherapy.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats.

BACKGROUND: Acellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI-NCSC-SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. METHODS: The effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI-NCSC-SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. RESULTS: (1) EPI-NCSC-SCLCs had good compatibility with ANAs in vitro. (2) In ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI-NCSC-SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). CONCLUSIONS: EPI-NCSC-SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI-NCSC-SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.

ASH2L mediates epidermal differentiation and hair follicle morphogenesis via H3K4me3 modification.

The processes of epidermal development in mammals are regulated by complex molecular mechanisms, such as histone modifications. Histone H3 lysine K4 (H3K4) methylation mediated by COMPASS methyltransferase is associated with gene activation, but its effect on epidermal lineage development remains unclear. Therefore, we constructed a mouse model of specific ASH2L (COMPASS methyltransferase core subunit) deletion in epidermal progenitor cells and investigated its effect on the development of mouse epidermal lineage. Furthermore, downstream target genes regulated by H3K4me3 were screened using RNA-sequencing combined with Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing. Deletion of ASH2L in epidermal progenitor cells caused thinning of the suprabasal layer of the epidermis and delayed hair follicle morphogenesis in newborn mice. These phenotypes may be related to the reduced proliferative capacity of epidermal and hair follicle progenitor cells. ASH2L depletion may also lead to depletion of the epidermal stem cell pools in late mouse development. Finally, genes related to hair follicle development (Shh, Edar and Fzd6), Notch signaling pathway (Notch2, Notch3, Hes5 and Nrarp) and ΔNp63 were identified as downstream target genes regulated by H3K4me3. Collectively, ASH2L-dependent H3K4me3 modification served as an upstream epigenetic regulator in epidermal differentiation and hair follicle morphogenesis in mice.

Approaches to Study Wound-Induced Hair Neogenesis (WIHN).

Embryonic wound repair proceeds with complete regeneration of the tissue without any scar formation, whereas tissue repair in adults usually results in scars and the tissue does not completely regain its preinjured state. Wound-induced hair neogenesis (WIHN) in adult rodents results in de novo hair follicle formation in the center of large wounds, mimicking regeneration processes seen in fetal tissue. The investigation of WIHN therefore provides a unique quantitative framework for scrutinizing the mechanistic underpinnings of regenerative repair, which can have clinical implications in the context of scarless healing. In this chapter, we present a detailed protocol for inducing wounds that lead to hair neogenesis in laboratory mice and facilitating the identification and characterization of distinct stages in neogenic hair follicle development. Additionally, we present a whole-mount alkaline phosphatase assay to distinguish de novo hair follicles. These protocols can facilitate studies toward obtaining a comprehensive understanding of WIHN and shedding light on the intricate molecular and cellular processes involved in mammalian regenerative repair.

Modulating embryonic signaling pathways paves the way for regeneration in wound healing.

Epithelial tissues, including the skin, are highly proliferative tissues with the capability to constant renewal and regeneration, a feature that is essential for survival as the skin forms a protective barrier against external insults and water loss. In adult mammalian skin, every injury will lead to a scar. The scar tissue that is produced to seal the wound efficiently is usually rigid and lacks elasticity and the skin's original resilience to external impacts, but also secondary appendages such as hair follicles and sebaceous glands. While it was long thought that hair follicles develop solely during embryogenesis, it is becoming increasingly clear that hair follicles can also regenerate within a wound. The ability of the skin to induce hair neogenesis following injury however declines with age. As fetal and neonatal skin have the remarkable capacity to heal without scarring, the recapitulation of a neonatal state has been a primary target of recent regenerative research. In this review we highlight how modulating dermal signaling or the abundance of specific fibroblast subsets could be utilized to induce de novo hair follicles within the wound bed, and thus to shift wound repair with a scar to scarless regeneration.

Recent progress in hair follicle stem cell markers and their regulatory roles.

Hair follicle stem cells (HFSCs) in the bulge are a multipotent adult stem cell population. They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing. An increasing number of biomarkers have been used to isolate, label, and trace HFSCs in recent years. Considering more detailed data from single-cell transcriptomics technology, we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

Follicular unit grafting in chronic ulcers: A valuable technique for integrated management.

Chronic ulcers significantly affect the quality of life of patients and impose a high cost on the healthcare system. The therapeutic management should be comprehensive, taking into consideration the etiological diagnosis of the wound and the characteristics of the wound bed when deciding on a therapeutic proposal appropriate to the healing phase, correcting factors that delay healing. During the epithelialization phase, repair techniques with grafts are recommended to shorten re-epithelialization time, improve the quality of scar tissue, and achieve adequate pain management. Currently, due to the reported benefits of skin appendages, the technique of follicular unit auto-grafting obtained with a scalp punch is among the chosen strategies for wound repair. This is a minimally invasive, outpatient practice, whose technique has advantages over the donor site, patients recovery and well-being.

Acquired curved hair is caused by fusion of multiple hair matrix cells.

BACKGROUND: "Curved hair" caused by acquired factors is considered to have adverse cosmetic effects, but the detailed mechanism behind curved hair remains obscure. OBJECTIVE: We attempted to clarify the causes of curved hair that appeared to have occurred via acquired factors. METHODS: Outer root sheath cells (ORSC) isolated from plucked human hair follicles were used to evaluate the expression of type IV collagen. Straight and curved hairs with hair follicle tissue attached were also collected from the same individuals and subjected to morphological, immunohistochemical, and gene expression analyses. RESULTS: The amount of type IV collagen increased upon inducing endoplasmic reticulum stress in ORSC. Meanwhile, in curved hair follicle tissue, the gene expression of type IV collagen decreased. In addition, the curved hair follicle tissue obtained from participants in their 30 s to 50 s had distorted shapes compared with that of straight hair from the same individuals. It was also observed that hair matrix cells based on multiple hair germs fused to eventually form a single hair follicle and hair shaft. In curved hair follicle tissue, KRT71 protein, a marker of inner root sheath differentiation, was unevenly distributed and there was elevated expression of Dickkopf-1 (DKK1) protein, an inhibitor of the Wnt signaling pathway. CONCLUSION: Our study revealed the fusion of hair matrix cells during hair follicle regeneration as a cause of acquired curved hair. We consider that such fusion causes hair follicle tissue to abnormally differentiate, resulting in asymmetric hair follicle shapes and curved hair.

Translational frontiers: insight from lymphatics in skin regeneration.

The remarkable regenerative ability of the skin, governed by complex molecular mechanisms, offers profound insights into the skin repair processes and the pathogenesis of various dermatological conditions. This understanding, derived from studies in human skin and various model systems, has not only deepened our knowledge of skin regeneration but also facilitated the development of skin substitutes in clinical practice. Recent research highlights the crucial role of lymphatic vessels in skin regeneration. Traditionally associated with fluid dynamics and immune modulation, these vessels are now recognized for interacting with skin stem cells and coordinating regeneration. This Mini Review provides an overview of recent advancements in basic and translational research related to skin regeneration, focusing on the dynamic interplay between lymphatic vessels and skin biology. Key highlights include the critical role of stem cell-lymphatic vessel crosstalk in orchestrating skin regeneration, emerging translational approaches, and their implications for skin diseases. Additionally, the review identifies research gaps and proposes potential future directions, underscoring the significance of this rapidly evolving research arena.

The promising prospect of human hair follicle regeneration in the shadow of new tissue engineering strategies.

Hair loss disorder (alopecia) affects numerous people around the world. The low effectiveness and numerous side effects of common treatments have prompted researchers to investigate alternative and effective solutions. Hair follicle (HF) bioengineering is the knowledge of using hair-inductive (trichogenic) cells. Most bioengineering-based approaches focus on regenerating folliculogenesis through manipulation of regulators of physical/molecular properties in the HF niche. Despite the high potential of cell therapy, no cell product has been produced for effective treatment in the field of hair regeneration. This problem shows the challenges in the functionality of cultured human hair cells. To achieve this goal, research and development of new and practical approaches, technologies and biomaterials are needed. Based on recent advances in the field, this review evaluates emerging HF bioengineering strategies and the future prospects for the field of tissue engineering and successful HF regeneration.

Screening of hair follicle telogen-associated circRNAs in sheep and construction of their ceRNA network.

Sheep breeds with hair-shedding traits have many advantages over non-shedding sheep breeds, not only because of reduced shearing labor and feeding management costs but also because it reduces in vitro parasites and improves adaptability to summer heat stress. The wool of Dorper sheep naturally sheds in spring due to the periodic growth of hair follicles. CircRNAs primarily regulate the morphogenesis of hair follicles through the ceRNA mechanism. In this study, five 2-year-old Dorper ewes with extreme hair-shedding phenotype (S) and three Dorper ewes with non-shedding (N) phenotype were selected for subsequent analyses. For RNA extraction, skin tissues were collected on 27th September 2019 (S1, N1), 3rd January 2020 (S2, N2), and 17th March 2020 (S3, N3), which were then subjected to RNA-seq. RNA-seq technology revealed 20,185 novel circRNAs in the hair follicles of Dorper sheep. Among them, 1450 circRNAs were differentially expressed (DE). Clustering heatmap and expression pattern analyses were performed on DE circRNAs, which indicated 78 circRNAs with T pattern (Telogen, highly expressed in telogen), and the source genes for candidate circRNAs were further screened by functional enrichment analysis, which identified 13 crucial genes enriched in pathways associated with hair follicle development. Additionally, a ceRNA regulatory network comprising 4 circRNAs, 11 miRNAs, and 13 target genes was constructed. Overall, this study screened circRNAs that may be associated with the telogen phase of hair follicles in sheep, providing a relevant theoretical basis for wool shedding in sheep and for breeding Dorper sheep with automatic wool shedding.

TLR2 regulates hair follicle cycle and regeneration via BMP signaling.

The etiology of hair loss remains enigmatic, and current remedies remain inadequate. Transcriptome analysis of aging hair follicles uncovered changes in immune pathways, including Toll-like receptors (TLRs). Our findings demonstrate that the maintenance of hair follicle homeostasis and the regeneration capacity after damage depend on TLR2 in hair follicle stem cells (HFSCs). In healthy hair follicles, TLR2 is expressed in a cycle-dependent manner and governs HFSCs activation by countering inhibitory BMP signaling. Hair follicles in aging and obesity exhibit a decrease in both TLR2 and its endogenous ligand carboxyethylpyrrole (CEP), a metabolite of polyunsaturated fatty acids. Administration of CEP stimulates hair regeneration through a TLR2-dependent mechanism. These results establish a novel connection between TLR2-mediated innate immunity and HFSC activation, which is pivotal to hair follicle health and the prevention of hair loss and provide new avenues for therapeutic intervention.

MiR-23b and miR-133 Cotarget TGFß2/NOTCH1 in Sheep Dermal Fibroblasts, Affecting Hair Follicle Development.

Wool is produced and controlled by hair follicles (HFs). However, little is known about the mechanisms involved in HF development and regulation. Sheep dermal fibroblasts (SDFs) play a key role in the initial stage of HF development. Analyzing the molecular mechanism that regulates early HF development in superfine wool sheep is of great importance for better understanding the HF morphogenesis process and for the breeding of fine wool sheep. Here, we show that two microRNAs (miRNAs) affect the development of HFs by targeting two genes that are expressed by SDFs. Meanwhile, the overexpression and inhibition of oar-miR-23b and oar-miR-133 in SDFs cells and cell proliferation, apoptosis, and migration were further detected using a CCK-8 assay, an Annexin V-FITC assay, a Transwell assay, and flow cytometry. We found that oar-miR-23b, oar-miR-133, and their cotarget genes TGFß2 and NOTCH1 were differentially expressed during the six stages of HF development in superfine wool sheep. Oar-miR-23b and oar-miR-133 inhibited the proliferation and migration of SDFs and promoted the apoptosis of SDFs through TGFß2 and NOTCH1. oar-miR-23b and oar-miR-133 inhibited the proliferation and migration of SDFs by jointly targeting TGFß2 and NOTCH1, thereby inhibiting the development of superfine wool HFs. Our research provides a molecular marker that can be used to guide the breeding of ultrafine wool sheep.

Hair follicle stem cells promote epidermal regeneration under expanded condition.

Skin soft tissue expansion is the process of obtaining excess skin mixed with skin development, wound healing, and mechanical stretching. Previous studies have reported that tissue expansion significantly induces epidermal proliferation throughout the skin. However, the mechanisms underlying epidermal regeneration during skin soft tissue expansion are yet to be clarified. Hair follicle stem cells (HFSCs) have been recognized as a promising approach for epidermal regeneration. This study examines HFSC-related epidermal regeneration mechanisms under expanded condition and proposes a potential method for its cellular and molecular regulation.