Epidermal Stem Cells Research Articles

Characterization of the Newborn Epidermis and Adult Hair Follicles Using Whole-Mount Immunofluorescent Staining of Mouse Dorsal Skin.

The mammalian integumentary system, including skin and its appendages, serves as a protective barrier for the body. During development, skin epidermis undergoes rapid cell division and differentiation to form multiple stratified layers of keratinocytes. Concurrently the epidermis also gives rise to hair follicles that invaginate into the dermis. In adult skin, the hair follicle undergoes cyclic regeneration fueled by hair follicle stem cells located in the bulge. Three-dimensional and high-resolution imaging of these structures using whole-mount immunofluorescent staining allows to better characterize epidermal progenitors and stem cells.

Emerging Perspectives of YAP/TAZ in Human Skin Epidermal and Dermal Aging.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are key downstream effectors of the Hippo signaling pathway, which plays a central role in tissue homeostasis, organ development, and regeneration. While the dysregulation of YAP/TAZ has been linked to various human diseases, their involvement in the aging of human skin has only recently begun to manifest. In the skin, the YAP/TAZ effectors emerge as central regulators in maintaining homeostasis of epidermal stem cells and dermal extracellular matrix, and thus intimately linked to skin aging processes. This review underscores recent molecular breakthroughs highlighting how age-related decline of YAP/TAZ activity impacts human epidermal and dermal aging. Gaining insight into the evolving roles of YAP/TAZ in human skin aging presents a promising avenue for the development of innovative therapeutic approaches aimed at enhancing skin health and addressing age-related skin conditions.

Isolation, Characterization, and Utilization of Human Skin Basal and Suprabasal Epidermal Stem Cells.

Studying human skin biology can aid in comprehending the pathophysiology of skin diseases and developing novel cell-based therapies, including tissue engineering approaches. This chapter provides a comprehensive guide of methods to determine human skin samples from the perspective of their cellular compositions. We describe as useful technique the histological analysis of tissue sections. We further illustrate the biological characterization of isolated and cultured basal and suprabasal interfollicular keratinocytes by cell sorting, cytospin immunostaining, colony forming efficiency, and long-term dermo-epidermal organotypic cultures.

Effect of Retinoic Acid on HaCaT and NIH-3T3 cells in an in vitro 3D Collagen Cell Culture Skin Model

Human skin aging is characterized by epidermal and dermal thinning, loss of elasticity, and wrinkles. Keratinocytes, the most common type of skin cell and fibroblasts, present in the cellular stroma beneath the skin’s surface, each play a role in aging. Using these cell types in in vitro research can reveal a deeper understanding of the dermatological function and cellular changes in aged skin. 3D cell culture techniques provide an opportunity to use these cell types in a model that can more accurately mimic human skin. Treatment of aging skin is of interest to both medical and consumer communities. Retinoic acid (RA) is a metabolite of vitamin A and retinol that assists in cell proliferation, differentiation, and immune functions. Over the counter (OTC) and prescription retinoids are common topical products used for anti-aging and acne treatments. This study seeks to determine the impact of topical retinoid creams on keratinocyte proliferation and morphology in 3D cell culture models of aged and unaged human skin. NIH-3T3 fibroblasts were embedded in a 3D collagen matrix of varying thickness, and HaCaT keratinocytes were seeded on top of the matrix at varying seeding densities to mimic aging and youthful skin. 0.1 µM 0.025% tretinoin and 0.1 µM 0.1% adapalene topical creams were prepared in culture medium and used to treat cells daily, on alternate days, or just once during a week-long period. Alamar Blue assays and microscopy showed that tretinoin treatment was cytotoxic at this concentration, with a single treatment reducing cell viability by ~43% compared to the untreated control. Adapalene treatment, while showing significantly greater cell proliferation than tretinoin, did not exceed the proliferation of the untreated control. It is understood that retinol increases cell turnover by killing cells rapidly, so it is proposed that in our model, the rate of proliferation does not overcome the rate of cell death. Cell viability trends remain similar between young and old skin model treatments. Future studies should focus on creating a 3D model more accurately representing the aging in vivo skin environment where keratinocytes can be readily differentiated from epidermal stem cells. KEYWORDS: HaCaTs; NIH-3T3; Retinoids; Retinoic Acid; Tretinoin; Adapalene; 3D Collagen Gel; Proliferation

Combination Therapy with Platelet-Rich Plasma and Epidermal Neural Crest Stem Cells Increases Treatment Efficacy in Vascular Dementia.

This study aimed to evaluate the efficacy and treatment mechanism of platelet-rich plasma (PRP) and neural crest-derived epidermal stem cells (ESCs) in their administration alone and combination in vascular dementia (VaD) model by two-vessel occlusion (2VO). Methods. Sixty-six rats were divided into six groups: the control, sham, 2VO + vehicle, 2VO + PRP, 2VO + ESC, and 2VO + ESC + PRP. The treated groups received 1 million cells on days 4, 14, and 21 with or without 500 µl PRP (twice a week) after 2VO. The memory performance and anxiety were evaluated by behavioral tests including open field, passive avoidance, and Morris water maze. The basal-synaptic transmission (BST) and long-term potentiation (LTP) were assessed through field-potential recordings of the CA1. The mRNA expression levels of IGF-1, TGF-β1, PSD-95, and GSk-3β were measured in the rat hippocampus by quantitative reverse transcription polymerase chain reaction. Results. The results demonstrated impaired learning, memory, and synaptic plasticity in the 2VO rats, along with a significant decrease in the expression of IGF-1, TGF-β1, PSD-95, and upregulation of GSK-3β. Treatment with ESC alone and ESC + PRP showed similar improvements in spatial memory and LTP induction, with associated upregulation of PSD-95 and downregulation of GSK-3β. However, only the ESC + PRP group showed recovery in BST. Furthermore, combination therapy was more effective than PRP monotherapy for LTP and memory. Conclusions. The transplantation of ESC showed better effects than PRP alone, and combination therapy increased the treatment efficacy with the recovery of BST. This finding may be a clue for the combination therapy of ESC and PRP for VaD.

Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells

Epidermolysis Bullosa Simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most of EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient’s keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon1. Through the CRISPR/Cas9 system we performed a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Effect of Metformin on Epidermal Neural Crest Stem Cells and Their Potential Application in Ameliorating Paclitaxel-induced Neurotoxicity Phenotype.

Epidermal Neural Crest Stem Cells (EPI-NCSCs) have emerged as prospective ideal candidates to meet the fundamental requirements of cell-based therapies in neurodegenerative disorders. The present study aimed to identify the potential of metformin in driving EPI-NCSCs to neuronal/glial differentiation and express neurotrophic factors as well as assess their therapeutic potential for mitigating the main behavioral manifestations of chemotherapy-induced neurotoxicity (CIN). EPI-NCSCs were extracted from the bulge region of hair follicle. Following expansion, transcript and protein expression profiles of key markers for stemness (Nestin, EGR-1, SOX-2 and 10), neurotrophic activity (BDNF, GDNF, NGF, FGF-2, and IL-6), and neuronal (TUB3, DCX, NRF and NeuN) and glial (PDGFRα, NG2, GFAP, and MBP) differentiation were determined on days 1 and 7 post-treatment with 10 and 100μM metformin using real time-PCR and immunocytochemistry methods. Then, the in vivo function of metformin-treated stem cells was evaluated in the context of paclitaxel CIN. To do so, thermal hyperalgesia, mechanical allodynia, and spatial learning and memory tests were evaluated by Hotplate, Von Frey, and Morris water maze tests. Our result indicated that exposure of EPI-NCSCs to metformin was associated with progressive decline in stemness markers and enhanced expression levels of several neurotrophic, neuron and oligodendrocyte-specific markers. Further, it was observed that intranasal metformin-treated EPI-NCSCs improved the cognitive impairment, and mechanical and thermal hypersensitivity induced by paclitaxel in rats. Collectively, we reasoned that metformin pretreatment of EPI-NCSCs might further enhance their therapeutic benefits against CIN.

Human Skin Aging and the Anti-Aging Properties of Retinol.

The skin is the most-extensive and -abundant tissue in the human body. Like many organs, as we age, human skin experiences gradual atrophy in both the epidermis and dermis. This can be primarily attributed to the diminishing population of epidermal stem cells and the reduction in collagen, which is the primary structural protein in the human body. The alterations occurring in the epidermis and dermis due to the aging process result in disruptions to the structure and functionality of the skin. This creates a microenvironment conducive to age-related skin conditions such as a compromised skin barrier, slowed wound healing, and the onset of skin cancer. This review emphasizes the recent molecular discoveries related to skin aging and evaluates preventive approaches, such as the use of topical retinoids. Topical retinoids have demonstrated promise in enhancing skin texture, diminishing fine lines, and augmenting the thickness of both the epidermal and dermal layers.

A cosmopolitan Serendipita forms mycothalli with sub-Antarctic leafy liverworts

The occurrence of mycothalli, symbioses between liverworts and fungi bearing similarities to mycorrhizas, is poorly documented in sub-Antarctica, and biogeographical patterns in Serendipita, one of the main fungal genera forming the symbiosis, remain understudied. Here, 83 specimens of 16 leafy liverwort species in eight families were hence sampled from 12 sites on the Thatcher Peninsula on sub-Antarctic South Georgia and were examined for mycothalli. Staining and epifluorescence microscopy were used to enumerate fungal structures in liverwort tissues, and PCR amplification and sequencing of fungal ribosomal DNA were used to determine the taxonomic and biogeographical affinities of the fungi present in plants. Stained hyphal coils, a defining feature of the symbiosis, were found to be frequent (>40 % of stem length colonised) in Barbilophozia hatcheri (Anastrophyllaceae), Cephaloziella varians (Cephaloziellaceae) and Lophoziopsis excisa (Lophoziaceae). Analyses of fungal ITS2 region sequences showed that a single species of Serendipita, based on a 3 % cut-off for sequence divergence, was a frequent colonist of B. hatcheri, C. varians and L. excisa, and that a further 18 basidiomycete and ascomycete taxa colonised other liverwort species. Principal component analysis and analysis of variance indicated that the presence of the Serendipita species was positively associated with the occurrence of stained hyphal coils in stem epidermal cells. Phylogenetic analyses, incorporating worldwide accessions from leafy liverwort-associated Serendipita, showed that the same species, which also occurs in Chile, mainland Europe and on Svalbard, is apparently the sole symbiont of sub- and maritime Antarctic leafy liverworts, and indicated much higher species richness of the genus outside Antarctica.

METTL14-mediated N6-methyladenosine modification of Col17a1/Itgα6/Itgβ4 governs epidermal homeostasis

BackgroundN6-methyladenosine (m6A) is the most abundant and reversible modification occurring in eukaryotic mRNAs, however, its functions in mammalian epidermal development are still not fully elucidated. ObjectiveTo explore the role of METTL14 (Methyltransferase like 14), one of the m6A methyltransferases, in maintaining epidermal homeostasis. MethodsWe constructed mice with Mettl14-inactivation in the epidermal basal cells. The phenotype was explored by H&E staining and immunofluorescence staining. To explore the underlying mechanisms, we performed RNA-seq, Ribosome profiling and MeRIP-seq on wild-type and Mettl14-inactivation epidermal keratinocytes. Moreover, HaCaT cells were used for in vitro validation. ResultsInactivation of Mettl14 in murine epidermis led to transient thicker epidermis and exhaustion of the epidermal stem cell pool. Interestingly, we found that the mRNA of type XVII collagen (Col17a1), integrin β4 (Itgβ4) and α6 (Itgα6) had m6A modifications, and the proteins expression were decreased in Mettl14-inactivated epidermis. Furthermore, in epidermis-specific Mettl4-inactivated mice, the epidermis was detached from the dermis and presented a phenotype similar to junctional epidermolysis bullosa (JEB), which may result from hemidesmosomes damage (decrease of COL17A1, ITGB4 and ITGA6). Knockdown of Mettl14 in HaCaT cells impaired the self-renewal and decreased the protein level of COL17A1, ITGB4 and ITGA6 and Itgβ4 knockdown inhibited colony formation. ConclusionOur study highlighted the role of METTL14 in the maintenance of epidermal homeostasis and identified its critical role through m6A-mediated translational inhibition of Col17a1, Itgβ4 and Itgα6. Our study suggested that METTL14 may be a potential therapeutic target for the treatment of hemidesmosomes-deficient diseases, such as JEB.

Photobiomodulation Facilitates Rat Cutaneous Wound Healing by Promoting Epidermal Stem Cells and Hair Follicle Stem Cells Proliferation.

Cutaneous wound healing represents a common fundamental phenomenon requiring the participation ofcells of distinct types and a major concern for the public. Evidence has confirmed that photobiomodulation (PBM) usingnear-infrared (NIR)can promote wound healing, but the cells involved and theprecise molecular mechanisms remain elusive. Full-thickness skin defects with a diameter of 1.0cm were made on the back of rats and randomly divided into the control group, 10J, 15J, and 30J groups. Thewound healing rate at days 4, 8, and 12 postoperativelywasmeasured.HE and Masson staining was conducted to revealthe histological characteristics. Immunofluorescence staining was performedto label the epidermal stem cells (ESCs) and hair follicle stem cells (HFSCs). Western blot was performed to detect the expressions ofproteins associated withESCs and HFSCs. Cutaneous wound tissues were collected for RNA sequencing. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analysis was performed, and the hub geneswereidentified usingCytoHubba and validated by qRT-PCR. PBM can promote reepithelialization, extracellular matrix deposition, and wound healing, increase the number of KRT14+/PCNA+ ESCs and KRT15+/PCNA+ HFSCs, and upregulate the protein expression of P63, Krt14, and PCNA. Three hundred and sixty-six differentially expressed genes (DEGs) and 7hubgenes includingSox9, Krt5, Epcam, Cdh1, Cdh3, Dsp, and Pkp3were identified. These DEGs are enriched in skin development, cell junction, and cadherin binding involved in cell-cell adhesion etc., while these hub genes are related toskin derived stem cells and cell adhesion. PBM accelerates wound healing by enhancing reepithelialization through promoting ESCs and HFSCs proliferation and elevating the expression of genes associated with stem cells and cell adhesion. This may provide a valuable alternative strategy to promote wound healing and reepithelialization by modulating the proliferation ofskin derived stem cells and regulating genes related to cell adhesion.

Epidermal Stem Cell Derived Exosomes Alleviate Excessive Autophagy Induced Endothelial Cell Apoptosis by Delivering miR200b-3p to Diabetic Wounds

The dysfunction of endothelial cells caused by hyperglycemia is observed as a decrease in neovascularization in diabetic wound healing. Studies have found that epidermal stem cells (EpiSCs) can promote the angiogenesis of full-thickness wounds. To further explain the therapeutic effect of EpiSCs, EpiSC-derived exosomes (EpiSC-EXOs) are considered the main substance contributing to stem cell effectivity. In our study, EpiSCs and EpiSC-EXOs were supplied to the dorsal wounds of db/db mice. Results showed that EpiSCs could colonize in the wound area and both EpiSCs and EpiSC-EXOs could accelerate diabetic wound healing by promoting angiogenesis. Invitro, persistent high glucose led to the malfunction and apoptosis of endothelial cells. The apoptosis induced by high glucose is due to excessive autophagy and was alleviated by EpiSC-EXOs. RNA sequencing of EpiSC-EXOs showed that miR200b-3p was enriched in EpiSC-EXOs and alleviated the apoptosis of endothelial cells. Synapse defective rho GTPase homolog 1 was identified the target of miR200b-3p and affected the phosphorylation of ERK to regulate intracellular autophagy and apoptosis. Furthermore, animal experiments validated the angiogenic effect of miR200b-3p. Collectively, our results verified the effect of EpiSC-EXOs on apoptosis caused by hyperglycemia in endothelial cells through the miR200b-3p/synapse defective rho GTPase homolog 1 /RAS/ERK/autophagy pathway, providing a theoretical basis for EpiSC in treating diabetic wounds.

Constructing epidermal rete ridges using a composite hydrogel to enhance multiple signaling pathways for the maintenance of epidermal stem cell niche.

The undulating microstructure rete ridge (RR) located at the junction between the dermis and epidermis plays a crucial role in improving skin mechanical properties and maintaining skin homeostasis. However, the investigation of RR microstructures is usually neglected in current tissue engineering for skin regeneration. Here, to create an epidermal model with RR microstructures, keratinocytes were cultured on a patterned GelMA-PEGDA hydrogel constructed using molding technology. Furthermore, grafting acryloylated Arg-Gly-Asp (RGD) peptides on the hydrogel surface significantly improved cell adhesion, fusion, and development. RT-PCR, Western blot, and immunofluorescence staining confirmed that cells on RR microstructures exhibited higher gene and protein expression associated with epidermal stem cells. RNA sequencing analysis of cells on RR microstructure showed higher gene expression profiles related to stem cell maintenance, basement membrane formation, and epidermal development. Furthermore, RT-PCR analysis of epidermal models of various dimensions demonstrated that smaller microstructures were more conducive to epidermal stem cell marker gene expression, which is analogous to human skin. Overall, we have successfully developed a method for integrating RR microstructures into an epidermal model that mimics natural skin to maintain epidermal stem cell niche, providing a valuable reference for researching skin regeneration within the fields of tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This study presents a method for precisely fabricating microstructures of skin rete ridges using composite hydrogels, thereby creating a skin model that mimics natural human skin. The findings reveal that this microstructure provides a stem cell niche that regulates the pathways and promotes the expression of proteins related to epidermal stem cells. This work advances the functional properties of tissue engineered skin and holds promise for improving the therapeutic efficacy of artificial skin grafts for the skin wounds.

Myc-dependent dedifferentiation of Gata6+ epidermal cells resembles reversal of terminal differentiation

Dedifferentiation is the process by which terminally differentiated cells acquire the properties of stem cells. During mouse skin wound healing, the differentiated Gata6-lineage positive cells of the sebaceous duct are able to dedifferentiate. Here we have integrated lineage tracing and single-cell mRNA sequencing to uncover the underlying mechanism. Gata6-lineage positive and negative epidermal stem cells in wounds are transcriptionally indistinguishable. Furthermore, in contrast to reprogramming of induced pluripotent stem cells, the same genes are expressed in the epidermal dedifferentiation and differentiation trajectories, indicating that dedifferentiation does not involve adoption of a new cell state. We demonstrate that dedifferentiation is not only induced by wounding, but also by retinoic acid treatment or mechanical expansion of the epidermis. In all three cases, dedifferentiation is dependent on the master transcription factor c-Myc. Mechanotransduction and actin-cytoskeleton remodelling are key features of dedifferentiation. Our study elucidates the molecular basis of epidermal dedifferentiation, which may be generally applicable to adult tissues.

Matricellular Proteins in the Homeostasis, Regeneration, and Aging of Skin.

Matricellular proteins are secreted extracellular proteins that bear no primary structural functions but play crucial roles in tissue remodeling during development, homeostasis, and aging. Despite their low expression after birth, matricellular proteins within skin compartments support the structural function of many extracellular matrix proteins, such as collagens. In this review, we summarize the function of matricellular proteins in skin stem cell niches that influence stem cells' fate and self-renewal ability. In the epidermal stem cell niche, fibulin 7 promotes epidermal stem cells' heterogeneity and fitness into old age, and the transforming growth factor-β-induced protein ig-h3 (TGFBI)-enhances epidermal stem cell growth and wound healing. In the hair follicle stem cell niche, matricellular proteins such as periostin, tenascin C, SPARC, fibulin 1, CCN2, and R-Spondin 2 and 3 modulate stem cell activity during the hair cycle and may stabilize arrector pili muscle attachment to the hair follicle during piloerections (goosebumps). In skin wound healing, matricellular proteins are upregulated, and their functions have been examined in various gain-and-loss-of-function studies. However, much remains unknown concerning whether these proteins modulate skin stem cell behavior, plasticity, or cell-cell communications during wound healing and aging, leaving a new avenue for future studies.

Y-27632 acts beyond ROCK inhibition to maintain epidermal stem-like cells in culture.

Conditional reprogramming is a cell culture technique that effectively immortalizes epithelial cells with normal genotypes by renewing epidermal stem cells. Y-27632, a compound that promotes conditional reprogramming through an unknown mechanism, was developed to inhibit the two Rho-associated kinase (ROCK) isoforms. We used human foreskin keratinocytes (HFKs) to study the role of Y-27632 in conditional reprogramming and learn how ROCKs control epidermal stem cell renewal. In conditional reprogramming, Y-27632 increased HFK adherence to culture dishes, progression through S, G2 and M phases of the cell cycle, and epidermal stem cell marker levels. Although this correlated with ROCK inhibition by Y-27632, we generated CRISPR-Cas9-mediated HFK ROCK knockouts to test the direct role of ROCK inhibition. Knockout of single ROCK isoforms was insufficient to disrupt ROCK activity or promote HFK propagation without Y-27632. Although ROCK activity was reduced, HFKs with double knockout of ROCK1 and ROCK2 still required Y-27632 to propagate. Y-27632 was the most effective among the ROCK inhibitors we tested at promoting HFK proliferation and epidermal stem cell marker expression. Thus, the ability of Y-27632 to promote an epidermal stem cell state in conditional reprogramming not only depends upon ROCK inhibition but also acts via as-yet-unidentified mechanisms. Epidermal stem cell renewal might in part be regulated by ROCKs, but also involves additional pathways.

Physiological Mechanism of Waterlogging Stress on Yield of Waxy Maize at the Jointing Stage.

In the main agricultural area for waxy maize production in China, waterlogging occurs frequently during the waxy maize jointing stage, and this causes significant yield reduction. It is very important to understand the physiological mechanism of waterlogging stress in waxy maize during the jointing stage to develop strategies against waterlogging stress. Therefore, this study set waterlogging treatments in the field for 0, 2, 4, 6, 8, and 10 days during the waxy maize jointing stage, and were labelled CK, WS2, WS4, WS6, WS8 and WS10, respectively. By analyzing the effect of waterlogging on the source, sink, and transport of photoassimilates, the physiological mechanism of waterlogging stress in the jointing stage was clarified. The results show that PEPC and POD activities and Pro content decreased significantly under WS2 compared to CK. Except for these three indicators, the Pn, GS, leaf area, kernel number, yield, and puncture strength of stems were significantly decreased under the WS4. Under the WS6, the content of MDA began to increase significantly, while almost all other physiological indices decreased significantly. Moreover, the structure of stem epidermal cells and the vascular bundle were deformed after 6 days of waterlogging. Therefore, the threshold value of waterlogging stress occured at 4 to 6 days in the jointing stage of waxy maize. Moreover, waterlogging stress at the jointing stage mainly reduces the yield by reducing the number of kernels; specifically, the kernel number decreased by 6.7-15.5% in 4-10 days of waterlogging, resulting in a decrease of 9.9-20.2% in the final yield. Thus, we have shown that waterlogging stress at the jointing stage results in the decrease of potential waxy maize kernel numbers and yield when the synthesis of sources was limited and the transport of photoassimilates was restricted.

Genome-wide association studies in the Japanese population identified genetic loci and target gene associated with epidermal turnover.

The epidermis is an essential organ for life by retaining water and as a protective barrier. The epidermis is maintained through metabolism, in which basal cells produced from epidermal stem cells differentiate into spinous cells, granular cells and corneocytes, and are finally shed from the epidermal surface. This is epidermal turnover, and with aging, there is a decline in epidermis function. Other factors that may affect epidermal turnover include ultraviolet damage and genetic factors. These genetic factors are of particular interest as little is known. Although recent skin-focused genome-wide association studies (GWAS) have been conducted, the genetic regions associated with epidermal turnover are almost uninvestigated. Therefore, we conducted a GWAS on epidermal turnover in the Japanese population, using the corneocyte area, which correlates to the rate of epidermal turnover, as an indicator. As a result, rs2278431 (p = 1.29 × 10-7 ) in 19q13.2 was associated with corneocyte size. Furthermore, eQTL analysis suggested that rs2278431 was related to the SPINT2 gene. In addition, SPINT2 knockdown studies using epidermal keratinocytes revealed that SPINT2 is involved in keratinocyte proliferation and in corneocyte size regulation in reconstructed epidermis. These results suggest that rs2278431 is involved in the expression of SPINT2 and affects epidermal turnover.

ΔNp63 Regulates Homeostasis, Stemness, and Suppression of Inflammation in the Adult Epidermis

The p63 transcription factor is critical for epidermis formation in embryonic development, but its role in the adult epidermis is poorly understood. In this study, we show that acute genetic ablation of ΔNp63, the main p63 isoform, in adult epidermis disrupts keratinocyte proliferation and self-maintenance and, unexpectedly, triggers an inflammatory psoriasis-like condition. Mechanistically, single-cell RNA sequencing revealed the downregulation of cell cycle genes, upregulation of differentiation markers, and induction of several proinflammatory pathways in ΔNp63-ablated keratinocytes. Intriguingly, ΔNp63-ablated cells disappear by 3 weeks after ablation, at the expense of the remaining nonablated cells. This is not associated with active cell death and is likely due to reduced self-maintenance and enhanced differentiation. Indeed, invivo wound healing, a physiological readout of the epidermal stem cell function, is severely impaired upon ΔNp63 ablation. We found that the Wnt signaling pathway (Wnt10A, Fzd6, Fzd10) and the activator protein 1 (JunB, Fos, FosB) factors are the likely ΔNp63 effectors responsible for keratinocyte proliferation/stemness and suppression of differentiation, respectively, whereas IL-1a, IL-18, IL-24, and IL-36γ are the likely negative effectors responsible for suppression of inflammation. These data establish ΔNp63 as a critical node that coordinates epidermal homeostasis, stemness, and suppression of inflammation, upstream of known regulatory pathways.

Mechanism of wound repair in diabetic rats using nanosilver-free alginate dressing.

Nanosilver-alginate dressing can effectively promote the healing of diabetic wounds in rats. However, due to the potential toxicity of nanosilver, its widespread application in hard-to-heal wound healing is limited. In the present study, the role and potential mechanism of nanosilver-free alginate gel (NSFAG) in the healing process of diabetic wounds were explored. A diabetic rat skin wound model was established, and wounds were treated with saline (NC group), nanosilver gel (NSG group) or nanosilver-free alginate gel (NSFAG group) for seven consecutive days. NSFAG significantly promoted wound healing and increased the content of protein and hydroxyproline in granulation tissues, and was superior to NSG (p<0.05). Immunohistochemical analyses revealed that the skin wound tissue structure of the NSFAG group was intact, and the number of skin appendages in the dermis layer was significantly higher compared with the NC group and the NSG group (p<0.05). Western blot analysis found that the protein expression of the epidermal stem cell marker molecules CK19 and CK14 as well the proliferation marker of keratinocytes Ki67 in the NSFAG group was significantly higher compared with the NC group or NSG group (p<0.05). Additionally, the proliferation marker of keratinocytes Ki67 in the NSFAG group was significantly higher compared with the NC or NSG group (p<0.05). Immunofluorescence staining analyses indicated that the CK19- and CK14-positive cells were mainly distributed around the epidermis and the newly formed appendages in the NSFAG group, and this result was not observed in the NC or NSG groups. The present findings demonstrate that NSFAG can effectively accelerate wound healing in diabetic rats by promoting epidermal stem cell proliferation and differentiation into skin cells, as well as formation of granulation tissue, suggesting that it can be a potential dressing for diabetic wounds.