Mosaicism in Cutaneous Disorders.

Genetic mosaicism arises when a zygote harbors two or more distinct genotypes, typically due to de novo, somatic mutation during embryogenesis. The clinical manifestations largely depend on the differentiation status of the mutated cell; earlier mutations target pluripotent cells and generate more widespread disease affecting multiple organ systems. If gonadal tissue is spared-as in somatic genomic mosaicism-the mutation and its effects are limited to the proband, whereas mosaicism also affecting the gametes, such as germline or gonosomal mosaicism, is transmissible. Mosaicism is easily appreciated in cutaneous disorders, as phenotypically distinct mutant cells often give rise to lesions in patterns determined by the affected cell type. Genetic investigation of cutaneous mosaic disorders has identified pathways central to disease pathogenesis, revealing novel therapeutic targets. In this review, we discuss examples of cutaneous mosaicism, approaches to gene discovery in these disorders, and insights into molecular pathobiology that have potential for clinical translation.

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Treating epidermolytic ichthyosis and ichthyosis with confetti with epidermal autografts cultured from revertant skin.

BACKGROUND: No efficient treatment has yet been established for epidermolytic ichthyosis (EI), which is caused by pathogenic variants of KRT1 or KRT10. Patients with ichthyosis with confetti (IWC) have multiple normal-appearing spots, caused by the revertant somatic recombination of pathogenic variants that occurs at each spot independently. Additionally, some patients with EI have large areas of normal skin due to revertant postzygotic mosaicism. OBJECTIVES: To assess the feasibility of transplanting cultured epidermal autografts (CEAs) produced from revertant epidermal keratinocytes in patients with EI and IWC. METHODS: We performed a clinical trial of treatment with CEAs produced from each patient's own revertant epidermal keratinocytes as a proof-of-concept study. This was a single-arm, open, unmasked, uncontrolled, single-assignment, treatment-purpose study. The primary outcome was the percentage area that lacked recurrence of ichthyosis lesions 4 weeks after the final transplant. The secondary outcome was the percentage area lacking recurrence of ichthyosis lesions 24 weeks after the initial transplantation. The trial was registered with the Japan Registry of Clinical Trials (jRCTb041190097). RESULTS: We successfully produced CEAs from genetically confirmed revertant skin from two patients with mosaic EI and from one patient with IWC and confirmed by amplicon sequencing and droplet digital polymerase chain reaction analysis that the CEAs mainly consisted of revertant wild-type cells. Single-cell RNA sequencing analysis confirmed the normal proliferation and safety profiling of CEAs. CEAs were transplanted onto desquamated lesional sites in the patients. Four weeks post-transplantation, the percentage area lacking recurrence of ichthyosis lesions in the three patients was 40%, 100% and 100% respectively, although recurrence of ichthyosis lesions was seen at the site of CEA transplantation in all three patients at 24 weeks post-transplantation. CONCLUSIONS: CEAs from normal skin have the potential to be a safe and local treatment option for EI and IWC.

Epidermolytic ichthyosis is a rare skin condition that causes redness, blistering and thickening of the skin. There is currently no effective treatment for the disease, which is caused by mutations in the genes KRT1 or KRT10. People with a type of the disease called 'ichthyosis with confetti' have many normal-appearing spots that are caused by the natural repair of the gene mutations. Some people with epidermolytic ichthyosis have large areas of healthy skin as a result of genetic mutations having been corrected. In this study, we successfully produced skin grafts from the healthy skin of two patients with epidermolytic ichthyosis and one with 'ichthyosis with confetti'. We confirmed that the skin grafts mainly consisted of repaired skin cells. A technique called 'single-cell RNA sequencing' confirmed the skin cells in the skin grafts behaved like healthy skin cells and that the grafts were safe. Overall, our study findings suggest that skin grafts taken from skin consisting of genetically normal keratinocytes that have undergone self-repair have potential to be a safe treatment option for patients with severe epidermolytic ichthyosis and 'ichthyosis with confetti'.

Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly.

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.

Nonsense mutations in KRT1 caused recessive epidermolytic palmoplantar keratoderma with knuckle pads.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is characterized by diffuse hyperkeratosis affecting palms and soles with suprabasal epidermolysis or vacuolar degeneration histopathologically. The disorder is caused by heterozygous mutations in KRT9 or KRT1. Dominant-negative mutations in KRT1 could also result in epidermolytic ichthyosis with EPPK, a more severe entity affecting the entire body. OBJECTIVE: To investigate the genetic basis and pathogenesis of two unrelated patients with EPPK and knuckle pads, both of whom were born to consanguineous parents of Chinese origin. METHODS: Next-generation sequencing was applied to the two patients using genomic DNA extracted from peripheral blood. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western blot (WB) were employed to evaluate mRNA and protein expression level. Ultrastructural changes of skin lesion were analysed using transmission electron microscopy. RESULTS: Two novel homozygous mutations, c.457C>T (p.Gln153*) and c.33C>G (p.Tyr11*) in KRT1, were identified in patients 1 and 2 respectively. The nonsense mutations were predicted to result in nonsense-mediated mRNA decay and absence of keratin 1, which was confirmed in the skin lesions from patient 1. Upregulated keratin 2 was detected both in the affected and unaffected skin samples from patient 1, while the protein abundance and distribution pattern of keratin 10 remained unchanged. An aberrant and clumped staining pattern of keratin 9 was noted in the palmar skin of patient 1. CONCLUSIONS: Homozygous 'knockout' mutations in KRT1 resulted in EPPK with knuckle pads rather than epidermolytic ichthyosis. We speculated that sparing of non-acral skin might be due to compensatory effect of keratin 2 upregulation by forming heterodimer with keratin 10.

Transcription Factors Runx1 and Runx3 Suppress Keratin Expression in Undifferentiated Keratinocytes.

The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.

Post Zygotic, Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer, Producing a Monolateral Palmar Epidermolytic Nevus.

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63-mediated transcription of KRT10 to improve psoriasis.

Psoriasis is a chronic immune-mediated inflammatory dermatosis. Recently, ozone therapy has been applicated to psoriasis treatment; however, the mechanism by which ozone therapy improves psoriasis remains unclear. The excessive proliferation and the differentiation of basal keratinocytes have been considered critical issues during pathological psoriasis process, in which keratin 6 (KRT6) and KRT10 might be involved. In the present study, KRT6, IL-17 and IL-22 protein within psoriasis lesions was decreased, while KRT10 and Tp63 protein in psoriasis lesions was increased by ozone treatment in both patient and IMQ mice psoriatic tissues. In the meantime, ozone treatment down-regulated KRT6 mRNA and protein expression while up-regulated KRT10 mRNA and protein expression within IL-22 treated primary KCs; the cell viability of KCs was suppressed by ozone treatment. Moreover, Tp63 bound to KRT10 promoter region to activate its transcription in basal keratinocytes; the promotive effects of ozone on Tp63 and KRT10 were significantly reversed by Tp63 silence. Both TP63 and KRT10 mRNA expression were significantly increased by ozone treatment in psoriasis lesions; there was a positive correlation between Tp63 and KRT10 expression within tissue samples, suggesting that ozone induces the expression of Tp63 to enhance the expression of KRT10 and the differentiation of keratinocytes, therefore improving the psoriasis. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of keratinocytes via increasing Tp63-mediated transcription of KRT10, therefore improving psoriasis.

Mutations in KRT10 in epidermolytic acanthoma.

BACKGROUND: Epidermolytic acanthoma (EA) is a rare acquired lesion demonstrating a characteristic histopathological pattern of epidermal degeneration referred to as epidermolytic hyperkeratosis (EHK). On histopathological analysis, EA appears nearly identical to inherited EHK-associated dermatoses such as epidermolytic ichthyosis and ichthyosis bullosa of Siemens. While it has been speculated that EA is caused by mutations in KRT10, KRT1, or KRT2 found in these inherited dermatoses, none have yet been identified. Herein, we aim to identify the contributions of keratin mutations to EA. METHODS: Using genomic DNA extracted from paraffin-embedded samples from departmental archives, we evaluated a discovery cohort using whole-exome sequencing (WES) and assessed remaining samples using Sanger sequencing screening and restriction fragment length polymorphism (RFLP) analysis. RESULTS: DNA from 16/20 cases in our sample was of sufficient quality for polymerase chain reaction amplification. WES of genomic DNA from lesional tissue revealed KRT10 c.466C > T, p.Arg156Cys mutations in 2/3 samples submitted for examination. RFLP analysis of these samples as well as eight additional samples confirmed the mutations identified via WES and identified four additional cases with Arg156 mutations. In sum, 6/11 screened cases showed hotspot mutation in KRT10. CONCLUSIONS: Hotspot mutations in the Arg156 position of KRT10, known to cause epidermolytic ichthyosis, also underlie EA.

Arginine- but not alanine-rich carboxy-termini trigger nuclear translocation of mutant keratin 10 in ichthyosis with confetti.

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.

Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis.

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.

Nail abnormalities identified in an ageing study of 30 inbred mouse strains.

In a large-scale ageing study, 30 inbred mouse strains were systematically screened for histologic evidence of lesions in all organ systems. Ten strains were diagnosed with similar nail abnormalities. The highest frequency was noted in NON/ShiLtJ mice. Lesions identified fell into two main categories: acute to chronic penetration of the third phalangeal bone through the hyponychium with associated inflammation and bone remodelling or metaplasia of the nail matrix and nail bed associated with severe orthokeratotic hyperkeratosis replacing the nail plate. Penetration of the distal phalanx through the hyponychium appeared to be the initiating feature resulting in nail abnormalities. The accompanying acute to subacute inflammatory response was associated with osteolysis of the distal phalanx. Evaluation of young NON/ShiLtJ mice revealed that these lesions were not often found, or affected only one digit. The only other nail unit abnormality identified was sporadic subungual epidermoid inclusion cysts which closely resembled similar lesions in human patients. These abnormalities, being age-related developments, may have contributed to weight loss due to impacts upon feeding and should be a consideration for future research due to the potential to interact with other experimental factors in ageing studies using the affected strains of mice.

Increase of cystathionine-γ-lyase (CSE) during late wound repair: Hydrogen sulfide triggers cytokeratin 10 expression in keratinocytes.

The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-ß-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

Human neonatal stem cell-derived skin substitute improves healing of severe burn wounds in a rat model.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell-based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord-MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs-derived skin cells (hNSCs-SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta-derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs-SCs seeded in plasma significantly improved basic skin architecture, re-epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell-derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta-derived stem cells make them superlative candidates for the development of "off-the-shelf" artificial skin substitutes for future use.

Ginnalin B induces differentiation markers and modulates the proliferation/differentiation balance via the upregulation of NOTCH1 in human epidermal keratinocytes.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro-d-glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 µM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.

Immunohistochemical expression analysis of the human fetal lower urogenital tract.

We have studied the ontogeny of the developing human male and female urogenital tracts from 9 weeks (indifferent stage) to 16 weeks (advanced sex differentiation) of gestation by immunohistochemistry on mid-sagittal sections. Sixteen human fetal pelvises were serial sectioned in the sagittal plane and stained with antibodies to epithelial, muscle, nerve, proliferation and hormone receptor markers. Key findings are: (1) The corpus cavernosum in males and females extends into the glans penis and clitoris, respectively, during the ambisexual stage (9 weeks) and thus appears to be an androgen-independent event. (2) The entire human male (and female) urethra is endodermal in origin based on the presence of FOXA1, KRT 7, uroplakin, and the absence of KRT10 staining. The endoderm of the urethra interfaces with ectodermal epidermis at the site of the urethral meatus. (3) The surface epithelium of the verumontanum is endodermal in origin (FOXA1-positive) with a possible contribution of Pax2-positive epithelial cells implying additional input from the Wolffian duct epithelium. (4) Prostatic ducts arise from the endodermal (FOXA1-positive) urogenital sinus epithelium near the verumontanum. (5) Immunohistochemical staining of mid-sagittal and para-sagittal sections revealed the external anal sphincter, levator ani, bulbospongiosus muscle and the anatomic relationships between these developing skeletal muscles and organs of the male and female reproductive tracts. Future studies of normal human developmental anatomy will lay the foundation for understanding congenital anomalies of the lower urogenital tract.

Characterization of a New Reconstructed Full Thickness Skin Model, T-Skin™, and its Application for Investigations of Anti-Aging Compounds.

BACKGROUND: We have characterized a new reconstructed full-thickness skin model, T-Skin™, compared to normal human skin (NHS) and evaluated its use in testing anti-aging compounds. METHODS: The structure and layer-specific markers were compared with NHS using histological and immunohistological staining. In anti-aging experiments, T-SkinTM was exposed to retinol (10 µM) or vitamin C (200 µM) for 5 days, followed by immunohistological staining evaluation. RESULTS: T-Skin™ exhibits a well stratified, differentiated and self-renewing epidermis with a dermal compartment of functional fibroblasts. Epidermal (cytokeratin 10, transglutaminase 1), dermo-epidermal junction (DEJ) (laminin 5, collagen-IV, collagen VII) and dermally-located (fibrillin 1, procollagen I) biomarkers were similar to those in NHS. Treatment of T-Skin™ with retinol decreased the expression of differentiation markers, cytokeratin 10 and transglutaminase 1 and increased the proliferation marker, Ki67, in epidermis basal-layer cells. Vitamin C increased the expression of DEJ components, collagen IV and VII and dermal procollagen 1. CONCLUSIONS: T-Skin™ exhibits structural and biomarker location characteristics similar to NHS. Responses of T-Skin™ to retinol and vitamin C treatment were consistent with those of their known anti-aging effects. T-Skin™ is a promising model to investigate responses of epidermal, DEJ and dermal regions to new skin anti-ageing compounds.

γ-Propoxy-Sulfo-Lichenan Induces In Vitro Cell Differentiation of Human Keratinocytes.

BACKGROUND: As non-cellulosic ß-d-glucans are known to exert wound-healing activity by triggering keratinocytes into cellular differentiation, the functionality of a semisynthetic lichenan-based polysaccharide on skin cell physiology was investigated. METHODS: γ-Propoxy-sulfo-lichenan (γ-PSL, molecular weight 52 kDa, ß-1,3/1,4-p-d-Glucose, degree of substitution 0.7) was prepared from lichenan. Differentiation of primary human keratinocytes was assayed by the protein analysis of differentiation specific markers and by gene expression analysis (qPCR). The gene array gave insight into the cell signaling induced by the polysaccharide. RESULTS: γ-PSL (1 to 100 µg/mL) triggered keratinocytes, in a concentration-dependent manner, into the terminal differentiation, as shown by the increased protein expression of cytokeratin 1 (KRT1). Time-dependent gene expression analysis proved differentiation-inducing effects, indicating strong and fast KRT1 gene expression, while KRT10 expression showed a maximum after 12 to 24 h, followed by downregulation to the basal level. Involucrin gene expression was only changed to a minor extent, which was similar to loricrin and transglutaminase. Gene array indicated the influence of γ-PSL on MAP kinase and TGF-ß mediated signaling towards keratinocyte differentiation. CONCLUSION: The propoxylated lichenan may improve wound healing by topical application to promote the terminal barrier formation of keratinocytes.

IL-36ß Promotes Inflammatory Activity and Inhibits Differentiation of Keratinocytes In Vitro.

Objective Psoriasis is an immune-mediated inflammatory disease. Despite advances in the study of its pathogenesis, the exact development mechanism of psoriasis remains to be fully elucidated. Hyperproliferative epidermis plays a crucial role in psoriasis. This study aimed to investigate the effects of interleukin-36ß (IL-36ß) on keratinocyte dysfunction in vitro. Methods Human keratinocyte cell lines, HaCaT cells, were treated with 0 (control), 50 or 100 ng/ml IL-36ß respectively for 24 h. Cell viability was determined with a cell counting kit-8 assay. Flow cytometry was used to assess the effects of IL-36ß on apoptosis and cell cycle distribution. Expressions of the differentiation markers, such as keratin 10 and involucrin, were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). Expressions of the inflammatory cytokines, IL-1ß and IL-6 were tested by ELISA. Results CCK8 assay showed the survival rate had no significant difference between the control and treated group (P > 0.05). Flow cytometry analysis showed cell cycle arrest at S phase in the IL-36ß-treated groups compared with the control group (P < 0.05). RT-qPCR verified the decreased mRNA expressions of keratin 10 and involucrin in the IL-36ß-treated groups compared with the negative control (P < 0.01). ELISA showed 100 ng/ml IL-36ß enhanced levels of IL-1ß and IL-6 in culture supernatants of HaCaT cells compared with the negative control (P < 0.05). Conclusion Taken together, these findings suggest that IL-36ß could induce cell cycle arrest at S phase, inhibit keratin 10 and involucrin expressions and promote inflammatory activity in HaCaT cell lines.

Regulation of mesenchymal signaling in palatal mucosa differentiation.

Epithelial differentiation is thought to be determined by mesenchymal components during embryogenesis. In mice, palatal mucosa showed the region-specific keratinization pattern along antero-posterior axis. However, developmental mechanisms involved in oral mucosa differentiation with fine tuning of keratinization are not elucidated yet. To reveal this developmental mechanism, first, we conducted tissue recombination assay of the palate at E16 for 2 days which revealed that epithelial differentiation with specific localization of CK10 is modulated by mesenchymal components. Based on the results, we propose that mesenchymal signaling would determine the presumptive fate of developing palatal epithelium in spatiotemporal manner. Genome-wide screening analysis using laser micro-dissection to collect spatiotemporal specific molecules between anterior and posterior palate suggested Meox2 in the posterior mesenchymal tissue to be a candidate regulator controlling epithelial differentiation. To examine the detailed spatiotemporal function of Meox2, we employed in vitro organ cultivation with the loss- and gain-of-function studies at E14.5 for 2 and 4 days, respectively. Our results suggest that posteriorly expressed Meox2 modulates non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.