Tunneling nanotubes (TNTs) are thin, actin-based intercellular conduits that enable long-range transfer of organelles and signaling cargo. Although widely reported across multiple cell types, their presence in human skin cells has not been well described. This article describes a standardized protocol to detect and characterize TNTs in vitro between human epidermal keratinocytes and dermal fibroblasts. The method involves preparing a co-culture of primary cells, gentle fixation to preserve fragile TNTs, membrane labeling with wheat germ agglutinin, F-actin staining with phalloidin, and systematic z-stack imaging by inverted confocal microscopy to distinguish TNTs suspended above the substratum from adherent filopodia. Optional immunostaining for α-tubulin allows assessment of microtubule incorporation. TNTs are defined by three features: thin, straight protrusions connecting two or more cells, the presence of F-actin, and continuity across cell pairs in serial optical sections. Representative results demonstrate TNTs linking dermal-dermal, epidermal-epidermal, and dermal-epidermal pairs, with variable cytoskeletal composition (F-actin alone or F-actin plus α-tubulin). Critical steps include gentle fixation, use of fresh reagents, and acquisition of sufficient z-planes to avoid misclassification, while common artifacts include TNT breakage and incomplete staining. Together, these optimized steps enable reproducible TNT detection in skin cell systems and offer a methodological basis for future investigation of TNT-mediated communication in skin biology and regeneration.

UV radiation (UVR) drives high mutational burdens, yet precursor melanocytes accumulate these mutations without triggering immune clearance. Here, we investigated whether melanocyte-intrinsic transcriptional program(s) underlie immune tolerance to mutations resulting from UVR exposure. In primary human melanocytes, expression of PD-L1 (CD274) was dependent on microphthalmia-associated transcription factor (MITF), a crucial regulator of melanocyte development and an intermediate in the UV-tanning pathway. MITF directly activated PD-L1 transcription by binding a conserved upstream enhancer containing functional E-box elements. MITF determined both baseline melanocytic PD-L1 expression in healthy skin and its induction following UVR, independent of interferon signaling. Melanocyte-restricted Pd-l1 deletion in mice triggered CD8+ T cell infiltration and depigmentation after long-term UVB exposure, recapitulating features of human vitiligo. PD-L1-deficient human induced pluripotent stem cell (iPSC)-derived melanocytes underwent increased apoptosis and were more susceptible than PD-L1-intact melanocytes to gp100-specific CD8+ T cell killing. Thus, a melanocyte-intrinsic MITF-PD-L1 tolerance program protects melanocytes from autoimmune destruction, potentially facilitating early immune evasion during melanoma development and conversely underlying the responsiveness of melanoma to PD-1/PD-L1 blockade.

Oral mucosal wounds heal faster and with minimal scarring compared to skin injuries, yet the underlying mechanisms remain poorly understood. To explore the cellular and molecular basis of this difference, we performed single-cell RNA sequencing (scRNA-seq) on paired, uninjured human oral mucosa and skin tissues from the same donors. This approach enabled the construction of a comprehensive single-cell transcriptomic atlas, facilitating direct comparison of cellular composition, gene expression profiles, and intercellular communication between the two tissues. Our analysis revealed distinct tissue-specific heterogeneity among keratinocytes, fibroblasts, immune cells, and endothelial cells. Oral keratinocytes exhibited signatures associated with proliferation and metabolic activity, while oral fibroblasts and immune cells expressed gene profiles suggestive of pro-regenerative and anti-fibrotic functions. Cell-cell communication analysis indicated that endothelial cells in oral mucosa participate in interactions that may promote rapid tissue remodeling. Although our data were derived from uninjured tissues, the identified differentially expressed genes and enriched pathways suggest potential regulatory networks that may underlie the distinct wound-healing behaviors of oral mucosa and skin. A subset of these genes was validated by RT-PCR in both autologous and allogeneic samples across different age and sex groups, confirming the robustness and reproducibility of our findings. This study provides the first single-cell transcriptomic comparison of intact human oral mucosa and skin under steady-state conditions, establishing a foundational atlas that reveals intrinsic tissue-specific features and identifies candidate targets for promoting scarless healing.

Psoriasis is driven by sustained epidermal inflammation tightly coupled to dysregulated redox homeostasis. Although current systemic therapies are effective, their long-term use is limited by safety concerns. Dental pulp stem cell–derived exosomes (DPSC-Exo) have emerged as promising immunomodulatory, cell-free therapeutics, yet their role in regulating epithelial redox–inflammatory balance remains undefined. The therapeutic effects of DPSC-Exo were evaluated using an imiquimod-induced mouse model of psoriasis, ex vivo human skin explants, and M5-stimulated primary human keratinocytes. Transcriptomic profiling, immunostaining, and gain- and loss-of-function analyses were performed to define the underlying molecular mechanisms. Topical administration of DPSC-Exo markedly reduced epidermal hyperplasia, neutrophil infiltration, angiogenesis, and expression of key psoriatic mediators, including IL-23 A, IL-17 A, and antimicrobial peptides. These effects were consistently reproduced in human skin explants and keratinocyte models. RNA-sequencing identified glutathione peroxidase 2 (GPX2), a key epithelial antioxidant enzyme, as a prominently upregulated target following DPSC-Exo treatment. Restoration of GPX2 suppressed NF-κB activation and downstream cytokine production, whereas GPX2 silencing abolished the protective effects of DPSC-Exo. Mechanistically, two exosomal microRNAs, miR-1246 and miR-17-3p, were required for GPX2 induction and mediated the majority of the observed anti-inflammatory responses. These findings identify a previously unrecognized miRNA–GPX2–NF-κB axis through which DPSC-Exo restore epithelial redox–inflammatory homeostasis. Our study supports DPSC-Exo as a promising cell-free therapeutic candidate for psoriasis and highlights epithelial redox regulation as a potentially targetable mechanism for inflammatory skin diseases.

Dental pulp stem cells (DPSCs) represent an accessible and clinically relevant source of mesenchymal stem cells, and their derived exosomes have emerged as promising bioactive vesicles for cell-free applications in tissue engineering and regenerative medicine. However, reproducible and standardized methods for isolating and functionally validating DPSC-derived exosomes remain limited, particularly with respect to variability introduced during cell expansion across passages. Here, we describe a comprehensive, standardized, kit-based workflow for the isolation and purification of exosomes from DPSC-conditioned medium that is compatible with routine laboratory practice. This method enables consistent recovery of intact vesicles suitable for morphological, molecular, and functional characterization using commonly available techniques. Exosome identity is assessed based on morphology, size distribution, and marker expression, while biological activity is evaluated using a defined in vitro anti-inflammatory assay. To examine passage-related effects, exosomes derived from different DPSC passages are compared using this functional readout, providing a practical framework for assessing consistency during cell expansion. By integrating isolation, purification, characterization, and functional validation into a single workflow, this protocol offers a reproducible and scalable approach for generating functionally validated DPSC-derived exosomes for biomaterials research and regenerative applications.

Specialized cell types layer cell-type-restricted proteins, metabolites, and organelles onto a shared foundation of core cellular processes. How this ubiquitous machinery generates lineage-specific outputs is central to both cell biology and therapeutic development. G-protein-coupled receptors (GPCRs) respond to receptor-restricted ligands to drive cell-type-specific transcription through cAMP-mediated activation of protein kinase A (PKA), which activates the transcription factor CREB1 through two parallel modes: direct phosphorylation at serine 133, and inhibition of salt-inducible kinases (SIK) that restrain the CRTC coactivators. How these modes integrate and contribute to signaling across cell types has remained unresolved, obscured by genetic redundancy and essentiality. Here we combine focused genetic analyses with a cross-lineage transcriptomic survey to dissect these parallel inputs. In Creb1 / Atf1 / Crem triple-knockout cells, a non-phosphorylatable mutant CREB1 S133A fully rescued endogenous target gene activation, while Crtc1/Crtc2/Crtc3 ablation abolished transcription even with intact CREB1 serine 133 phosphorylation. As part of this mechanism, we found the annotated repressor ICER can instead act as a positive regulator, substituting for full-length CREB1 paralogs to drive a feedforward loop. Across melanocytes, hepatocytes, osteocytes, macrophages, and neurons, SIK inhibition recapitulated cAMP-PKA-driven transcription across both shared and cell-type-specific gene expression programs, with neurons a notable exception. These results invert the canonical model, placing CRTC recruitment as the dominant driver of CREB1-mediated transcription across diverse lineages, reframing how cAMP-PKA signaling can be interpreted and therapeutically targeted.

Acne vulgaris is one of the most prevalent chronic inflammatory skin disorders, affecting a substantial proportion of adolescents and adults worldwide. While conventional treatments such as antibiotics, hormonal therapy, and retinoids have been widely used, they often come with limitations such as drug resistance, systemic side effects, or poor long-term adherence. Recent years have witnessed a surge in innovative acne therapies that target the underlying pathophysiology with greater specificity and fewer adverse effects. These include oral agents that inhibit metabolic enzymes critical for sebaceous gland function, microbiome-targeted approaches such as bacteriophage therapy, next-generation topical agents with anti-androgen or anti-inflammatory effects, non-invasive devices like sebum-targeting lasers, and advanced follicular drug delivery systems. Furthermore, artificial intelligence (AI) is increasingly being employed to personalize acne management. This review provides a comprehensive overview of these emerging strategies, critically evaluates their mechanisms and clinical potential, and discusses future directions for integrated and precision acne therapy.

Stem cell niches are dynamic microenvironments that regulate tissue homeostasis. Epidermal stem cells (EpiSC) preferentially localize to concave regions of epidermal rete ridges, which serve as primary niches for stem cell maintenance. EpiSC number and functional integrity decline during chronological aging. A defining feature of aged skin is epidermal atrophy, in which the prominent rete ridges present in young skin become flattened. Whether such topographical alterations influence EpiSC homeostasis and differentiation remains unclear. To address this, we generated anatomically accurate rete ridge structures using 3D bioprinting of collagen matrices as an ex vivo model and compared EpiSC cultured within concave topography to those maintained on a flat matrix resembling aged skin. Transcriptomic analysis revealed that concave niches promoted keratinocyte differentiation, marked by increased type I and II keratin gene expression and downregulation of cell cycle–associated genes. ATAC-seq identified topography-dependent chromatin accessibility changes enriched for transcription factors regulating epidermal differentiation, including upregulation of KLF4 and GRHL3 and downregulation of SOX9, HOXA1, and ETS1. Consistently, aged human skin showed reduced KLF4 and GRHL3 and increased SOX9 compared with young skin. Our findings demonstrate that concave niche topography imposes a spatially defined EpiSC microenvironment that promotes differentiation, alters cell cycle, and when perturbed, potentially contributes to the aging process. We conclude that spatial localization within rete ridge regions significantly affects epidermal progenitor stemness properties as fundamental differences in the physical microenvironment appear to influence cell fate decisions, thus, form shapes function of EpiSC.

Introduction: Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising target in cancer therapy. Metformin, a widely used antidiabetic drug, has shown potential to induce ferroptosis, offering novel insights into its anticancer mechanisms. Methods: This review summarizes current literature on the molecular mechanisms of metformin-induced ferroptosis, focusing on its regulation of iron metabolism, redox homeostasis, lipid peroxidation, and relevant signaling pathways across different cancer types. Results: Metformin modulates key ferroptosis regulators, including SLC7A11, GPX4, AMPK, NRF2, and ACSL4, thereby promoting intracellular ROS accumulation, GSH depletion, and enhanced lipid peroxidation. Through these effects, metformin enhances ferroptotic sensitivity in multiple cancers and synergizes with ferroptosis inducers, chemotherapy, or radiotherapy to overcome resistance. Discussion: Although metformin shows promise in targeting ferroptosis, several challenges remain, including the heterogeneity of tumor ferroptosis sensitivity, optimal dosing strategies, and the need for reliable biomarkers. Furthermore, the dual roles of some ferroptosis regulators in cancer biology warrant cautious interpretation. Continued research is required to optimize therapeutic combinations and validate preclinical findings in clinical settings. Conclusion: Metformin-induced ferroptosis represents a promising anticancer strategy. Its integration into combination therapies could enhance treatment efficacy and overcome resistance, supporting its clinical repurposing in oncology.

Conventional treatments for androgenetic alopecia (AGA) have limitations, motivating alternative delivery approaches. This protocol describes the fabrication, characterization, and application of 3BDO-loaded hyaluronic acid/chitosan (HA/CS) dissolvable microneedle (MN) patches in a C57BL/6 mouse hair-regrowth model. The method uses a two-step molding process in a polydimethylsiloxane (PDMS) mold to concentrate 3BDO in the needle tips while forming a drug-free HA/CS backing layer. The patch is characterized by scanning electron microscopy (SEM) imaging, mechanical testing, skin insertion and dissolution testing, in vitro release testing, antibacterial assays, cytotoxicity testing, and in vivo hair-regrowth assessment. Upon insertion, the MN tips dissolve, releasing 3BDO locally. In vivo evaluation showed that the 3BDO-loaded MN group had more uniform hair regrowth than the subcutaneous injection and control groups tested. This protocol also supports assessment of patch morphology, insertion performance, and local biocompatibility. This method provides a preclinical workflow to evaluate MN-mediated delivery of 3BDO for hair-regrowth applications.

Tunneling nanotubes (TNTs) are membranous structures that enable direct intercellular transfer of mitochondria, proteins, RNAs, and signaling molecules, playing key roles in tissue repair, immune coordination, and stress adaptation. Among their critical functions, TNT-mediated mitochondrial transfer rescues metabolically impaired cells, yet the regulatory mechanisms governing TNT formation and function remain incompletely understood. Recent studies highlight the Wnt signaling pathway—a conserved regulator of cell fate, polarity, and cytoskeletal remodeling—as a central modulator of TNT dynamics. Through its canonical (Wnt/β-catenin) and non-canonical (Wnt/PCP and Wnt/Ca2+) branches, Wnt signaling orchestrates actin filament organization, bundling, and turnover, all of which are essential for TNT biogenesis and stability. This review critically examines the mechanistic intersection between Wnt signaling and TNTs, with an emphasis on how Wnt-driven cytoskeletal remodeling supports intercellular connectivity. Beyond basic mechanistic insights, we also explore the physiological and pathological relevance of this crosstalk—including its roles in tissue regeneration, immune modulation, cancer progression, and neurodegeneration. While the Wnt–TNT axis offers therapeutic promise, its context-dependent effects demand careful consideration.

While melanoma cells often express a high burden of mutated proteins, the infiltration of reactive T cells rarely results in tumor-eradicating immunity. We discovered that large extracellular vesicles, known as melanosomes, secreted by melanoma cells are decorated with major histocompatibility complex (MHC) molecules that stimulate CD8+ T cells through their T cell receptor (TCR), causing T cell dysfunction and apoptosis. Immunopeptidomic and T cell receptor sequencing (TCR-seq) analyses revealed that these melanosomes carry MHC-bound tumor-associated antigens with higher affinity and immunogenicity, which compete with their tumor cell of origin for direct TCR-MHC interactions. Analysis of biopsies from melanoma patients confirmed that melanosomes trap infiltrating lymphocytes, induce partial activation, and decrease CD8+ T cell cytotoxicity. Inhibition of melanosome secretion in vivo significantly reduced tumor immune evasion. These findings suggest that MHC export protects melanoma from the cytotoxic effects of T cells. Our study highlights a novel immune evasion mechanism and proposes a therapeutic avenue to enhance tumor immunity.

Inflammatory skin diseases such as psoriasis, atopic dermatitis, vitiligo, and lupus erythematosus present persistent therapeutic challenges due to their chronic nature, immune dysregulation, and suboptimal treatment outcomes. Stem cell-derived exosomes have emerged as a promising cell-free therapeutic approach, capable of modulating immune responses, promoting tissue regeneration, and restoring skin homeostasis. This review provides a comprehensive overview of exosomes derived from various stem cell sources-including adipose tissue, bone marrow, umbilical cord, dental pulp, and induced pluripotent stem cells-and their roles in regulating immune cell function and facilitating skin repair. Particular emphasis is placed on biomaterial-assisted delivery strategies, such as hydrogels, nanoparticles, and engineered scaffolds, which enhance the stability, targeting, and retention of exosomes at inflammatory sites. Key immunomodulatory mechanisms, including Treg/Th17 balance, macrophage polarization, and dendritic cell regulation, are also discussed. Finally, we highlight emerging preclinical and clinical evidence supporting the integration of exosome therapy with bioactive materials as a next-generation strategy for treating inflammatory skin disorders with improved precision and durability.

Misfolding of aggregation-prone proteins underpins diseases known as proteinopathies. One of these proteins, alpha-synuclein, is a component of aggregates in neurodegenerative conditions such as Parkinson's disease. The melanosomal protein PMEL, which forms physiologic amyloid scaffold structures on which melanin is organized in melanosomes, similarly ectopically accumulates in the dermis in many forms of cutaneous hyperpigmentation. Here, we demonstrate in a wide range of common clinical pigmentary disorders, as well as in primary melanocyte and mouse models examined by molecular, proteomic, and electron microscopic tools, that melanocytic alpha-synuclein is a prominent component of intracellular protein aggregates bound to similar proteins as in Parkinson's disease, as well as melanized extracellular protein deposits. Using the Real Time Quaking-Induced Conversion Assay (RT-QuIC), we demonstrate that UV induces misfolded melanosomal proteins to self-propagate, augmenting this pathology in prion-like fashion. CUT&RUN chromatin profiling and single-cell RNA-seq demonstrate that melanocytes utilize microphthalmia-associated transcription factor (MITF)-regulated autophagy to counteract protein aggregation, identifying aggregate removal as a core function of tanning. In contrast to extracellular aggregation, impaired intracellular aggregate removal contributes to melanocyte senescence, which conversely exacerbates chronic hypopigmentation and photoaging-related discoloration. These findings identify melanosomal proteinopathy as a common contributor to melanocyte dysfunction and suggest aggregate-focused management approaches.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rhoassociated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serumfree keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Background Psoriasis is a chronic inflammatory skin disease characterized by a complex pathogenesis involving various types of cells and cytokines. Among those, the pro-inflammatory cytokine IL-23/IL-17A axis plays a crucial role in the development and rapid progression of psoriasis. Phenformin, a derivative of metformin and a member of the biguanide class of drugs, exhibits superior anti-inflammatory and anti-tumor efficacy compared to metformin. However, the potential role of phenformin in anti-psoriatic skin inflammation has not been explored. Methods In this study, we utilized a mouse model of psoriasis and an in vitro model using human keratinocytes to investigate whether phenformin can suppress psoriasis-like inflammatory responses. Results Our results demonstrate that the topical application of phenformin significantly inhibited acute skin inflammatory responses in the psoriasis mouse model induced by imiquimod (IMQ). Additionally, phenformin suppressed the expression of psoriasis-related cytokines IL-17, IL-23, IL-8, and S100A8/S100A9 in an in vitro psoriatic keratinocyte model induced by IMQ. Furthermore, we found that IMQ-induced psoriatic skin and IMQ-treated keratinocytes exhibited high expression of the c-Myc gene, which was downregulated by phenformin. The c-Myc inhibitor JQ1 similarly inhibited the psoriatic inflammatory response and the expression of psoriasis-related cytokines in both in vitro and in vivo models. Conclusion phenformin ameliorates the psoriasis-like inflammatory response by inhibiting c-Myc expression in keratinocytes, suggesting its potential as a topical drug for the treatment of psoriasis.

Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. Skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from non-plantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers, as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating non-healing wounds.

The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.