Epidermal Differentiation Markers Research Articles

Vδ1 T Cells Integrated in Full-Thickness Skin Equivalents: A Model for the Role of Human Skin–Resident γδT Cells

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

EpidermaQuant: Unsupervised Detection and Quantification of Epidermal Differentiation Markers on H-DAB-Stained Images of Reconstructed Human Epidermis.

The integrity of the reconstructed human epidermis generated in vitro can be assessed using histological analyses combined with immunohistochemical staining of keratinocyte differentiation markers. Technical differences during the preparation and capture of stained images may influence the outcome of computational methods. Due to the specific nature of the analyzed material, no annotated datasets or dedicated methods are publicly available. Using a dataset with 598 unannotated images showing cross-sections of in vitro reconstructed human epidermis stained with DAB-based immunohistochemistry reaction to visualize four different keratinocyte differentiation marker proteins (filaggrin, keratin 10, Ki67, HSPA2) and counterstained with hematoxylin, we developed an unsupervised method for the detection and quantification of immunohistochemical staining. The pipeline consists of the following steps: (i) color normalization; (ii) color deconvolution; (iii) morphological operations; (iv) automatic image rotation; and (v) clustering. The most effective combination of methods includes (i) Reinhard's normalization; (ii) Ruifrok and Johnston color-deconvolution method; (iii) proposed image-rotation method based on boundary distribution of image intensity; and (iv) k-means clustering. The results of the work should enhance the performance of quantitative analyses of protein markers in reconstructed human epidermis samples and enable the comparison of their spatial distribution between different experimental conditions.

Cornulin as a Key Diagnostic and Prognostic Biomarker in Cancers of the Squamous Epithelium.

The prevalence of squamous cell carcinoma is increasing, and efforts that aid in an early and accurate diagnosis are crucial to improve clinical outcomes for patients. Cornulin, a squamous epithelium-specific protein, has recently garnered attention due to its implications in the progression of squamous cell carcinoma developed in several tissues. As an epidermal differentiation marker, it is involved in skin anchoring, regulating cellular proliferation, and is a putative tumor suppressor. The physiologically healthy squamous epithelium displays a considerable level of Cornulin, whereas squamous cell carcinomas have marked downregulation, suggesting that Cornulin expression levels can be utilized for the early detection and follow-up on the progression of these types of cancer. Cornulin's expression patterns in cervical cancer have been examined, and findings support the stepwise downregulation of Cornulin levels that accompanies the progression to neoplasia in the cervix. Additional studies documented a similar trend in expression in other types of cancer, such as cutaneous, esophageal, and oropharyngeal squamous cell carcinomas. The consistent and predictable pattern of Cornulin expression across several squamous cell carcinomas and its correlation with key clinicopathological parameters make it a reliable biomarker for assessing the transformation and progression events in the squamous epithelium, thus potentially contributing to the early detection, definitive diagnosis, and more favorable prognosis for these cancer patients.

Role of mesenchymal stem cell conditioned medium on wound healing using a developed 3D skin model.

Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.

PH-Responsive Calcium Ions and Crocetin Releasing Hydrogel for Accelerating Skin Wound Healing.

The ideal and highly anticipated dressing for skin wounds should provide a moist environment, possess antibacterial properties, and ensure sustained drug release. In the present work, a hyaluronic acid-based hydrogel was formed by cross-linking crocetin and CaCO3@polyelectrolyte materials (CaCO3@PEM) microspheres with HA hydrogels via hydrogen bond and amido bonding (CaCO3@PEM@Cro@HA hydrogel, CPC@HA hydrogel). Moreover, the CPC@HA hydrogel had the capability of sustained, controlled release of calcium ions and crocetin via pH-sensitive and accelerated skin wound healing. The experiment results showed that the CPC@HA hydrogel exhibited porous network structures, stable physical properties, and had antibacterial properties and biocompatibility in vitro. In addition, the CPC@HA hydrogel covering on the skin wound could reduce inflammation and promote wound healing. The high expression of angiogenic cytokines (CD31) and epidermal terminal differentiation markers (Loricrin) of wound healing tissue suggested the CPC@HA hydrogel also had the function of promoting the remodeling of regenerated skin. Overall, CPC@HA hydrogel has promising potential for clinical applications in accelerating skin wound repair.

Improving stability of human three dimensional skin equivalents using plasma surface treatment.

In developing three-dimensional (3D) human skin equivalents (HSEs), preventing dermis and epidermis layer distortion due to the contraction of hydrogels by fibroblasts is a challenging issue. Previously, a fabrication method of HSEs was tested using a modified solid scaffold or a hydrogel matrix in combination with the natural polymer coated onto the tissue culture surface, but the obtained HSEs exhibited skin layer contraction and loss of the skin integrity and barrier functions. In this study, we investigated the method of HSE fabrication that enhances the stability of the skin model by using surface plasma treatment. The results showed that plasma treatment of the tissue culture surface prevented dermal layer shrinkage of HSEs, in contrast to the HSE fabrication using fibronectin coating. The HSEs from plasma-treated surface showed significantly higher transepithelial electrical resistance compared to the fibronectin-coated model. They also expressed markers of epidermal differentiation (keratin 10, keratin 14 and loricrin), epidermal tight junctions (claudin 1 and zonula occludens-1), and extracellular matrix proteins (collagen IV), and exhibited morphological characteristics of the primary human skins. Taken together, the use of plasma surface treatment significantly improves the stability of 3D HSEs with well-defined dermis and epidermis layers and enhanced skin integrity and the barrier functions.

The Secretion of Inflammatory Cytokines Triggered by TLR2 Through Calcium‐Dependent and Calcium‐Independent Pathways in Keratinocytes

Keratinocytes can be activated by Cutibacterium acnes, leading to the production of proinflammatory cytokines via toll‐like receptors (TLRs) 2 and 4. Although several studies have investigated keratinocytes, the mechanism of calcium‐mediated activation remains unclear. Herein, we investigated whether calcium influx via TLR2 and TLR4 stimulation was involved in cytokine secretion by keratinocytes in HaCaT cells. Although TLR2 stimulation by peptidoglycan (PGN) increased intracellular calcium influx, TLR4 stimulation by lipopolysaccharide (LPS) did not increase it, as analyzed using flow cytometry with the calcium indicator Fluo‐3. However, activation by either TLR2 or TLR4 ligands upregulated the intracellular calcium influx in THP‐1 monocytes. Additionally, the expression of major proinflammatory cytokines and chemokines, such as interleukin (IL)‐6, IL‐8, IL‐1α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and monocyte chemoattractant protein‐1 (MCP‐1), was significantly increased by TLR2 in HaCaT cells. Moreover, treatment with the intracellular calcium chelator, BAPTA‐AM, disrupted PGN‐mediated induction of IL‐6, IL‐8, and MCP‐1 production. Real‐time quantitative polymerase chain reaction (PCR) and western blotting revealed that TLR2 stimulation induced expression of the epidermal differentiation marker keratin 1. In conclusion, TLR2‐induced intracellular calcium influx plays a pivotal role in the secretion of proinflammatory cytokines, such as IL‐6 and MCP‐1, in keratinocytes. Moreover, the continuous influx of calcium via TLR2 activation leads to keratinization. In vitro studies using HaCaT cells provide basic research on the effect of TLR2‐induced calcium on C. acnes‐mediated inflammation in keratinocytes. These studies are limited in their ability to clinically predict what happens in human keratinocytes. Clinical studies on patients with acne, including three‐dimensional (3D) cultures of primary keratinocytes, are required to develop new diagnostic markers for determining the severity of acne vulgaris.

Biological effect of laser-assisted scar healing (LASH) on standardized human three-dimensional wound healing skin models using fractional non-ablative 1540 nm Er:Glass or 1550 nm diode lasers.

In postoperative wound healing after surgical operations or ablative laser treatments, recent studies suggest the timely use of non-ablative fractional laser treatments with the aim to improve wound healing and prevent pathological scar formation. However, the underlying molecular mechanisms are poorly understood. The aim of this study was to investigate the effects of laser-assisted scar healing (LASH) at the molecular level and to combine it with already established wound healing-promoting local treatments. We irradiated full-thickness 3D skin models with a fractional ablative Er:YAG laser to set standardized lesions to the epidermal and upper dermal layer. Subsequently, LASH was induced by irradiating the models with either a fractional non-ablative 1540 nm Er:Glass or 1550 nm diode laser. In addition, we tested the combination of non-ablative fractional laser treatment and topical aftercare with a dexpanthenol-containing ointment (DCO). Histological analysis revealed that models irradiated with the 1540 nm Er:Glass or 1550 nm diode laser exhibited accelerated but not complete wound closure after 16 h. In contrast, additional topical posttreatment with DCO resulted in complete wound closure. At gene expression level, both non-ablative laser systems showed similar effects on epidermal differentiation and mild anti-inflammatory properties. The additional posttreatment with DCO enhanced the wound-healing effects of LASH, especially the upregulation of epidermal differentiation markers and anti-inflammatory cytokines at the gene expression level. This in vitro study deciphers the biological effects of LASH with a fractional non-ablative 1540 nm Er:Glass or a 1550 nm diode laser in 3D skin models. These data help to better understand the biological properties of the LASH technique and is important to optimize its application.

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Laser Treatment of Hypertrophic Scar in a Porcine Model Induces Change to Epidermal Histoarchitecture That Correlates to Improved Epidermal Barrier Function.

Mechanisms and timing of hypertrophic scar (HTS) improvement with laser therapy are incompletely understood. Epidermal keratinocytes influence HTS through paracrine signaling, yet they are understudied compared to fibroblasts. It was hypothesized that fractional ablative CO2 laser scar revision (FLSR) would change the fibrotic histoarchitecture of the epidermis in HTS. Duroc pigs (n = 4 FLSR and n = 4 controls) were injured and allowed to form HTS. HTS and normal skin (NS) were assessed weekly by noninvasive skin probes measuring trans-epidermal water loss (TEWL) and biopsy collection. There were 4 weekly FLSR treatments. Immediate laser treatment began on day 49 postinjury (just after re-epithelialization), and early treatment began on day 77 postinjury. Punch biopsies from NS and HTS were processed and stained with H&E. Epidermal thickness and rete ridge ratios (RRR) were measured. Gene and protein expression of involucrin (IVL) and filaggrin (FIL) were examined through qRT-PCR and immunofluorescent (IF) staining. After treatment, peeling sheets of stratum corneum were apparent which were not present in the controls. TEWL was increased in HTS vs NS at day 49, indicating decreased barrier function (P = .05). In the immediate group, TEWL was significantly decreased at week 4 (P < .05). The early group was not significantly different from NS at the prelaser timepoint. After four sessions, the epidermal thickness was significantly increased in treated scars in both FLSR groups (immediate: P < .01 and early: P < .001, n = 8 scars). Early intervention significantly increased RRR (P < .05), and immediate treatment trended toward an increase. There was no increase in either epidermal thickness or RRR in the controls. In the immediate intervention group, there was increased IVL gene expression in HTS vs NS that decreased after FLSR. Eight scars had upregulated gene expression of IVL vs NS levels pretreatment (fold change [FC] > 1.5) compared to four scars at week 4. This was confirmed by IF where IVL staining decreased after FLSR. FIL gene expression trended towards a decrease in both interventions after treatment. Changes in epidermal HTS histoarchitecture and expression levels of epidermal differentiation markers were induced by FLSR. The timing of laser intervention contributed to differences in TEWL, epidermal thickness, and RRR. These data shed light on the putative mechanisms of improvement seen after FLSR treatment. Resolution of timing must be further explored to enhance efficacy. An increased understanding of the difference between the natural history of HTS improvement over time and interventional-induced changes will be critical to justifying the continued approved usage of this treatment.

Cornulin as a Prognosticator for Lymph Node Involvement in Cutaneous Squamous Cell Carcinoma.

Background Cornulin is an epidermal differentiation marker and a stress-related protein. Its expression patterns are likely to reflect the multi-step tumorigenesis process of cSCC, given its role as a tumor suppressor.The aim of this study is to evaluate the utility of Cornulin as a prognosticator for cutaneous squamous cell carcinoma (cSCC). Specifically, the correlation between Cornulin expression and the clinicopathological parameter of lymph node involvement (nodal status), which plays a major role in determining cSCC prognosis and recurrence. We predicted that Cornulin expression declines as cSCC tumors metastasize to regional lymph nodes. Methodology Tissue samples of cSCC lesions of variable nodal involvement status were stained using immunohistochemistry, and high-resolution images were acquired. Aperio ImageScope software (Leica Biosystems) equipped with a positive-pixel-counting algorithm was used to quantify the staining intensity. Subsequently, Cornulin immunoreactivity was calculated as a Histo-score (H-score) value, which is based on the staining intensity and the percentage of positively stained cells. Mean H-scores were compared between groups using an unpaired t-test. Results A significant inverse correlation was found between Cornulin expression levels and metastasis to the lymph nodes. Specifically, primary tumors with metastasis to regional lymph nodes (N1) exhibited 9.5-fold decrease in Cornulin immunoreactivity compared to the primary tumor samples without lymph node involvement (N0). Conclusion Cornulin was found to be significantly downregulated in primary tumors with lymph node metastases. Detection assays to measure Cornulin expression in cSCC primary tumors might aid in determining the nodal status in these patientsand possibly help determine cases of occult lymph node metastasis or micrometastasis. Future clinical studies are needed to help establish Cornulin's role in enhancing the predictive power of histopathological examination and improving survival rates for patients suffering from this type of skin cancer.

Characterization of Cornulin as a Molecular Biomarker for the Progression of Oral Squamous Cell Carcinoma.

Introduction It has been shown that the expression of the epidermal differentiation marker, Cornulin, declines with the progression of squamous cell carcinomas of several tissue types. Objectives This study aims to examine Cornulin expression at the cellular level in various cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. This can pave the way for the utilization of this novel biomarker as a diagnostic and prognostic indicator for oral cancer and help guide treatment options. Study design Western blotting was performed to measure Cornulin expression levels using standardized cell lysates from four different cell lines representing the successive steps of OSCC progression. Specifically, primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells were used to represent normal oral keratinocytes, DOKs, locally invasive OSCC cells, and metastatic OSCC cells, respectively. Results Cornulin expression was found to be downregulated with the progression from normal to premalignant to malignant cells. Quantitative analysis revealed that Cornulin is significantly downregulated by 3.4 folds in DOK cells, by 23.7 folds in SCC25 cells, and by 5.2 folds in Detroit 562 cells compared to normal gingival keratinocytes. Interestingly, Cornulin was upregulated by 4.5 folds in the metastatic Detroit 562 cell line compared to the locally invasive SCC25 cells. Conclusion Altogether, Cornulin proved to be differentially expressed at the cellular level in cell lines representative of the successive steps of OSCC progression. Specifically, we documented a gradual decrease in Cornulin expression with the progression from normal to premalignant to malignant cells. Notably, there is a significant increase in the expression of Cornulin in the metastatic cell line Detroit 562 compared to the locally invasive cell line SCC25, suggesting a possible correlation with the biological behavior and unique characteristics of these two different phenotypes.

Cornulin as a Potential Novel Biomarker for Cutaneous Squamous Cell Carcinoma.

Background This study aimed to evaluate the expression of an epidermal differentiation marker, cornulin, in cutaneous squamous cell carcinoma (cSCC). Cornulin has been found to be downregulated in various squamous cell carcinomas of other tissues; however, its expression in cSCC has never been studied. We predicted that cornulin expression in cSCC is reduced compared to the normal epidermis. Moreover, we hypothesized that an inverse relationship exists between cornulin expression and the loss of differentiation, as defined by histopathological grading of cSCC lesions. Methodology Samples of normal skin and cSCC lesions of variable histopathological grades were stained using immunohistochemistry. High-resolution tissue images were analyzed with Aperio ImageScope (Leica Biosystems) utilizing a positive-pixel-counting algorithm to quantify the staining intensity. Histo-score (H-score) was calculated based on staining intensity and percentage of positive cell staining. Mean H-scores were compared using an unpaired t-test. Results We documented cornulin expression in cSCC for the first time. Cornulin levels were downregulated by more than two-fold in cSCC compared to the normal epidermis. Additionally, we observed a 4.5-fold downregulation in cornulin expression in tumors with high histopathological grades when compared to low histopathological grade tumors. Conclusions Cornulin expression levels measured through immunohistochemistry staining can help distinguish among the different histopathological grades of cSCC. Therefore, we propose that cornulin detection can be an adjunct to pathological examination to evaluate the differentiation status of cSCC specimens. Longitudinal studies are needed to establish the utility of cornulin as a diagnostic and prognostic biomarker for cSCC.

Downregulation of Lhx2 Markedly Impairs Wound Healing in Mouse Fetus.

Multiple transitions occur in the healing ability of the skin during embryonic development in mice. Embryos up to embryonic day 13 (E13) regenerate completely without a scar after full-thickness wounding. Then, up to E16, dermal structures can be formed, including skin appendages such as hair follicles. However, after E17, wound healing becomes incomplete, and scar formation is triggered. Lhx2 regulates the switch between maintenance and activation of hair follicle stem cells, which are involved in wound healing. Therefore, we investigated the role of Lhx2 in fetal wound healing. Embryos of ICR mice were surgically wounded at E13, E15, and E17, and the expression of Lhx2 along with mitotic (Ki67 and p63) and epidermal differentiation (keratin-10 and loricrin) markers was analyzed. The effect of Lhx2 knockdown on wound healing was observed. Lhx2 expression was not noticed in E13 due to the absence of folliculogenesis but was evident in the epidermal basal layer of E15 and E17 and at the base of E17 wounds, along with Ki67 and p63 expression. Furthermore, Lhx2 knockdown in E15 markedly prolonged wound healing and promoted clear scar formation. Therefore, Lhx2 expression is involved in cell division associated with wound healing and may contribute to scar formation in late embryos.

614 Laser-treatment of Hypertrophic Scar Induces Change to Epidermal Histoarchitecture Correlating to Improved Epidermal Barrier Function

IntroductionMechanisms and timing of hypertrophic scar (HTS) improvement with laser therapy are incompletely understood. Epidermal keratinocytes influence HTS through paracrine signaling, yet they are understudied compared to fibroblasts. It was hypothesized that fractional ablative CO2 laser scar revision (FLSR) would change the fibrotic histoarchitecture of the epidermis in HTS.MethodsDuroc pigs (n=4 FLSR and n=4 controls) were injured and allowed to form HTS. HTS and normal skin (NS) were assessed weekly by non-invasive skin probes measuring trans-epidermal water loss (TEWL) and biopsy collection. There were 4 weekly FLSR treatments. Early laser treatment began on day 49, and late began on day 77. Punch biopsies from NS and HTS were processed and stained with H&E. Image J was used to obtain epidermal thickness and rete ridge ratios (RRR). Gene and protein expression of involucrin (IVL) was examined through qRT-PCR and immunofluorescent (IF) staining.ResultsAfter treatment, peeling sheets of stratum corneum were apparent which were not present in the controls. TEWL was increased in HTS vs. NS at day 49 indicating decreased barrier function (42.2±8.0 vs. 22.0±4.62g/m2h, p=0.05). In the early group, TEWL was significantly decreased at week 4 to 16.4±3.5 g/m2h (p< 0.05). The late group was not significantly altered from NS at the pre-laser timepoint (day 77=12.1±1.99 g/m2h). Hence, there was no decrease in TEWL post-FLSR. After 4 sessions, epidermal thickness was significantly increased in treated scars in both FLSR groups (early:pre=85.6±6.8 vs. week 4=115.2±12.0 µm, p< 0.01) and (late:pre=75.2±6.6 vs. week 4=125.7±12.0 µm p< 0.001, n=8 scars,). There was no increase in controls. Late intervention significantly increased RRR (pre=1.3±0.1 vs. week 4=1.9±0.1, n=8 scars, p< 0.05), and early treatment trended towards increase (pre=1.17±0.05 vs. week 4=1.4 + 0.1). There was no increase in controls. There was increased IVL gene expression in HTS vs. NS that decreased after FLSR. Eight scars had up-regulated gene expression of IVL vs. NS levels pre-treatment (FC >1.5) compared to 4 scars at week 4. This was confirmed by IF where IVL staining decreased at week 4.ConclusionsChanges in epidermal HTS histoarchitecture and expression levels of epidermal differentiation markers were induced by FLSR. The timing of laser intervention contributed to differences in TEWL, epidermal thickness, and RRR.

Three-Dimensional Skin Tissue Printing with Human Skin Cell Lines and Mouse Skin-Derived Epidermal and Dermal Cells.

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

Effect of estrogen/progesterone ratio on the differentiation and the barrier function of epidermal keratinocyte and three-dimensional cultured human epidermis

AimsEstrogen (E) and progesterone (P) are the major female hormones and are secreted with changing concentration ratios throughout the menstrual cycle. These hormones have been studied individually regarding their physiological function in the skin, but their concentration ratio (E/P) and its effect on the skin has not yet been investigated. The purpose of this study was to clarify the effect of the E/P ratio on skin barrier function. The menstrual cycle was divided into the menstrual, follicular, ovulation, and luteal phases. Materials and methodsThe E/P concentration ratios corresponding with each phase were added to a three-dimensional epidermal model or normal human epidermal keratinocytes for 5 days. Gene and protein expression levels of several markers of cell differentiation, including loricrin (LOR) and transglutaminase (TGase), were quantified by real-time PCR and western blotting, respectively. Transepidermal water loss (TEWL) of the three-dimensional epidermal model was measured, and ceramide content was quantified by thin-layer chromatography. Key findingsGene expression of the epidermal differentiation markers, LOR and TGase, increased when applying the concentration ratio of E/P associated with the menstrual and luteal phases. The LOR protein level decreased from menstrual to luteal phases, and the TGase protein level increased from menstrual to luteal phases. During the same phases, ceramide NS increased and TEWL decreased. SignificanceSkin barrier function was improved by culturing cells at specific E/P concentration ratios, which would, therefore, be considered beneficial for female skin. This suggests that dysregulated E/P concentration ratios may be the cause of certain skin problems.

Djnedd4L Is Required for Head Regeneration by Regulating Stem Cell Maintenance in Planarians.

SUMOylation and ubiquitylation are homologous processes catalyzed by homologous enzymes, and they are involved in nearly all aspects of eukaryotic biology. Planarians, which have the remarkable ability to regenerate their central nervous system (CNS), provide an excellent opportunity to investigate the molecular processes of CNS regeneration in vivo. In this study, we analyzed gene expression profiles during head regeneration with an RNA-seq-based screening approach and found that Djnedd4L and Djubc9 were required for head regeneration in planarians. RNA interference targeting of Djubc9 caused the phospho-H3 mitotic cells to decrease in quantity, or even become absent as a part of the Djubc9 RNAi phenotype, which also showed the collapse of the stem cell lineage along with the reduced expression of epidermal differentiation markers. Furthermore, we found that Djnedd4L RNAi induced increased cell division and promoted the premature differentiation during regeneration. Taken together, our findings show that Djubc9 and Djnedd4L are required for stem cell maintenance in the planarian Dugesia japonica, which helps to elucidate the role of SUMOylation and ubiquitylation in regulating the regeneration process.