Human Epidermal Cells Research Articles

Luteolin-7-glucoside Promotes Human Epidermal Stem Cell Proliferation by Upregulating β-Catenin, c-Myc, and Cyclin Expression.

Skin epidermal stem cells (EpSCs) play critical roles in skin homeostasis and the repair of skin injury. Luteolin-7-glucoside (L7G) has been reported to accelerate skin wound healing through its anti-inflammatory and antioxidative activity. But its effect on EpSCs is not clear. In the present study, we examined the effect of L7G on the proliferation of human EpSCs and explored the mechanisms involved. MTT assay showed that L7G promoted EpSC proliferation in a dose- and time-dependent manner. BrdU incorporation assay and Ki67 immunofluorescence staining confirmed the proproliferative effect of L7G on EpSCs. Cell cycle analysis showed that treatment of EpSCs with L7G decreased the cell number in the G1 phase and increased the cell number in the S phase. In addition, L7G significantly enhanced EpSC migration. Mechanistic studies showed that L7G significantly induced the expression of β-catenin and c-Myc, as well as cyclins D1, A2, and E1 which are critical for G1/S phase transition. L7G stimulated EpSC proliferation through β-catenin and c-Myc. We further examined the effect of L7G on EpSC proliferation in skin tissues by treatment of human skin explants with L7G and examined the number of EpSCs by immunohistochemical stain of EpSC markers α6 integrin and β1 integrin. We found that treatment of human skin tissue explants with L7G significantly increased the thickness of the epidermis and increased the numbers of α6 integrin-positive and β1 integrin-positive cells at the basal layer of the epidermis. Taken together, these results indicate that L7G promotes EpSC proliferation through upregulating β-catenin, c-Myc, and cyclin expression. L7G can be used to expand EpSCs for generating epidermal autografts and engineered skin equivalents.

UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/β-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/β-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

646-P: The Efficacy and Safety of Cold Atmospheric Plasma as a Novel Therapy for Diabetic Wound In Vivo and In Vitro

Aims: Cold atmospheric plasma (CAP) interacted with tissue and had potential effects on wound healing and tissue regeneration. The aim of this study is to investigate the efficacy and safety of CAP as a novel therapy for diabetic wounds in vitro and in vivo. Methods: The plasma consists of ionized helium gas that is produced by a high-voltage (8.5kV) and high-frequency (17kHz) power supply. Eight-week-old male db/db mice were treated with conventional wound dressing only (control group), additional 90’s CAP (low-dose group) or 180’s CAP (high-dose group) for 2 weeks, respectively. Skin samples around wound in 3, 7, 14 days and blood samples were collected and analyzed in three groups. We carried out in vitro analysis included scratch wound healing assays in immortalized human epidermal HaCaT cells. Results: After 14 days of treatment, CAP could obviously promote diabetic wound healing because of inflammation inhibition and angiogenesis increase. Wound- closure rates of two CAP groups were significantly faster than that of control group. The protein expression of IL-6, TNF-α, iNOS and SOD significantly decreased while the protein level of VEGF, TGF-β in two CAP groups significantly increased compared to those in control group (all p < 0.05). And such changes showed a good consistency with the change in mRNA level (all p < 0.05). In vitro, scratch wound healing assays showed that plasma treatment could effectively accelerate the wound healing within 3 minutes exposure (all p < 0.05). Additionally, there were no significant differences in histological observation and the level of serum ALT, AST, BUN, CREA and WBC among the three groups. Conclusions: CAP treatment for 3 min daily improves diabetic wound healing by inhibiting inflammation, reducing oxidative stress and enhancing angiogenesis without toxicity to liver and kidney. Disclosure R. He: None. Q. Li: None. M. Yu: None. T. Wang: None. H. Lu: None. J. Lu: None. W. Zhu: None. M. Luo: None. J. Zhang: None. H. Gao: None. W. Xing: None. D. Wang: None. F. Liu: None. Funding National Natural Science Fund of China; Shanghai Science & Technology; Shanghai Municipal Education Commission

Propofol suppresses proliferation, invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9.

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.

Prodrugs for Skin Delivery of Menahydroquinone-4, an Active Form of Vitamin K2(20), Could Overcome the Photoinstability and Phototoxicity of Vitamin K2(20).

The effective delivery of menahydroquinone-4 (MKH), an active form of menaquinone-4 (MK-4, vitamin K2(20)), to the skin is beneficial in the treatment of various skin pathologies. However, its delivery through the application of MK-4 to the skin is hampered due to the photoinstability and phototoxicity of MK-4. This study aimed to evaluate the potential of ester prodrugs of MKH for its delivery into the skin to avoid the abovementioned issues. The ester prodrugs, MKH 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG) and MKH 1,4-bis-hemisuccinate (MKH-SUC), were prepared using our previously reported methods. Photostability was determined under artificial sunlight and multi-wavelength light irradiation, phototoxicity was determined by intracellular ROS formation and cell viability of UVA-irradiated human epidermal keratinocyte cells (HaCaT), and delivery of MKH into HaCaT cells was assessed by measuring menaquinone-4 epoxide (MKO) levels. MKH prodrugs showed higher photostability than MK-4. Although MK-4 induced cellular ROS and reduced cell viability after UVA irradiation, MKH prodrugs did not affect either ROS generation or cell viability. MKH prodrugs enhanced intracellular MKO, indicating effective delivery of MKH and subsequent carboxylation activity. In conclusion, these MKH prodrugs show potential for the delivery of MKH into the skin without photoinstability and phototoxicity.

Design and construction of a recombinant lentiviral vector with specific tropism to human epidermal growth factor-overexpressed cancer cells: Developing a new retargeting system for lentivirus vectors.

Targeting of specific tissues and cells by viruses is one of the challenges faced by researchers. Lentiviral vectors (LVs) are one of the most promising gene delivery systems in cancer gene therapy. Therefore, we aimed to design a novel lentiviral delivery system that expresses anti- human epidermal growth factor 2 (HER2) designed anykrin repeat protein (DARPin) on the vector envelope to create a pseudotyped lentivirus for targeting HER2-positive cancer cells. A helper plasmid producing the viral vector envelope containing anti-HER2 DARPin-G3 was constructed. LV was produced by transfer vector containing green fluorescent protein (GFP) gene and helper plasmids in human embryonic kidney 293 cells. The human breast cancer cell lines SKBR3 (normal and with inhibited endocytosis) (HER2-positive) and MDA-MB-231 (HER2-negative) were transduced by the recombinant viral vector. The GFP-based transduction rate was determined by flow cytometry and fluorescence microscopy. The anti-HER2 DARPin concentration in DARPin-LVs was significantly higher than the envelope G glycoprotein of the vesicular stomatitis virus-LVs (non-anti-HER2 control) (p<0.0001). In flow cytometry assays, the percentage of transduction by recombinant LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p=0.0074) and MDA-MB-231 cells (p=0.0037). In fluorescence microscopy assays, the percentage of transduction by new LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p=0.0026) and MDA-MB-231 cells (p=0.0014). We constructed a new recombinant LV with a defect in cell entry directly, containing an anti-HER2 DARPin on the vector envelope with specific tropism to HER2 receptor on HER2-positive cancer cells. We assumed that this viral vector transduces cells via an endocytosis-dependent process.

Pterostilbene Activates the Nrf2-Dependent Antioxidant Response to Ameliorate Arsenic-Induced Intracellular Damage and Apoptosis in Human Keratinocytes.

The NF-E2 p45-related factor 2 (Nrf2), a transcription factor that regulates the cellular adaptive response to oxidative stress, is a target for limiting tissue damage from exposure to environmental toxins, including arsenic. In the current study, we determine whether Pterostilbene (Pts), as a potent activator of Nrf2, has a protective effect on arsenic-induced cytotoxicity and apoptosis in human keratinocytes. Human keratinocytes (HaCaT) or mouse epidermal cells (JB6) were pretreated with Pts for 24 h prior to arsenic treatment. Harvested cells were analyzed by MTT, DCFH-DA, commercial kits, Flow cytometry assay and western blot analysis. Our results demonstrated that Pts effectively regulated the viability in HaCaT and JB6 cells, decreased the reactive oxygen species (ROS) generation and lipid peroxidation (MDA), and improved the NaAsO2-induced depletion of superoxide dismutase (SOD). Moreover, Pts treatment further dramatically inhibited NaAsO2-induced apoptosis, specifically the mitochondrial mediation of apoptosis, which coincided with the effective recovery of NaAsO2-induced mitochondrial membrane potential (ΔΨm) depolarization and cytochrome c release from the mitochondria. Furthermore, arsenic-induced decrease of anti-apoptotic factor Bcl-2 and Bcl-xl, and increase of pro-apoptotic factor Bax and Bad, as well as survival signal related factor caspase 3 activation were reversed by Pts treatment. Further mechanistic studies confirmed that Pts increased antioxidant enzyme expression in a dose-dependent manner, which was related to Nrf2 nuclear translocation. In addition, the effects of Pts on NaAsO2-induced cell viability were largely weakened when Nrf2 was knocked down. Together, our results provide evidence for the use of Pts to activate the Nrf2 pathway to alleviate arsenic-induced dermal damage.

Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa

Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and α6β4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells invitro and invivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for exvivo gene therapy of JEB.

Blimp-1 transcriptionally induced by growth factors, TNF-α and TLR ligands inhibits keratinocyte migration and inflammation but enhances cell migration in squamous cell carcinoma

Abstract B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. The aim of the study was to investigate the regulation and functional link between EGFR and Blimp-1 in human epidermal keratinocytes and squamous cell carcinoma (SCC). We found that EGF, PMA, TNF-α, LPS (TLR4 activator) and polyIC (TLR3 activator) can upregulate the protein and mRNA levels of Blimp-1 in NHEK and/or HaCaT cells. This effect of EGF was dependent on PKC, p38, and ERK activation, and Syk is also involved in the action of PMA. Additionally, the stability of Blimp-1 protein was under the control of the proteasome and lysosome degradation systems. PMA, LPS and polyIC also can activate EGFR and Syk in HaCaT cells. In addition, Blimp-1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. On the other hand, EGF, TNF-α, TGF-β and PMA could also upregulate Blimp-1 expression in SCC, and EGFR activation by TNF-α and PMA was observed. Unlike the action of Blimp-1 in keratinocytes, Blimp-1 silencing reduces SCC cell migration either in the absence or presence of TNF-α and PMA. Taken together, Blimp-1 can be upregulated by several growth factors, innate immune receptors and PKC signaling pathway. Blimp-1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, but mediates cell migration in SCC.

Synthesis and Biological Evaluation of 4β-N-Acetylamino Substituted Podophyllotoxin Derivatives as Novel Anticancer Agents.

A series of novel podophyllotoxin derivatives obtained by 4β-N-acetylamino substitution at C-4 position was designed, synthesized, and evaluated for in vitro cytotoxicity against four human cancer cell lines (EC-9706, HeLA, T-24 and H460) and a normal human epidermal cell line (HaCaT). The cytotoxicity test indicated that most of the derivatives displayed potent anticancer activities. In particular, compound 12h showed high activity with IC50 values ranging from 1.2 to 22.8 μM, with much better cytotoxic activity than the control drug etoposide (IC50: 8.4 to 78.2 μM). Compound 12j exhibited a promising cytotoxicity and selectivity profile against T24 and HaCaT cell lines with IC50 values of 2.7 and 49.1 μM, respectively. Compound 12g displayed potent cytotoxicity against HeLA and T24 cells with low activity against HaCaT cells. According to the results of fluorescence-activated cell sorting (FACS) analysis, 12g induced cell cycle arrest in the G2/M phase accompanied by apoptosis in T24 and HeLA cells. Furthermore, the docking studies showed possible interactions between human DNA topoisomerase IIα and 12g. These results suggest that 12g merits further optimization and development as a new podophyllotoxin-derived lead compound.

829 Folic acid protects melanocytes from oxidative damage by Keap1-Nrf2 pathway

This study aims to investigate whether folic acid could protect human melanocyte against oxidative damage and to elucidate the underlying pharmacological mechanism. And we demonstrate that folic acid could protect melanocytes form oxidative damage. Folic acid is of great therapeutic potential in the treatment of vitiligo. Vitiligo is a common skin disease characterized by the loss of functional melanocytes. Previous studies have indicated that oxidative stress plays a pivotal role in the onset of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Folic acid is a conventional antioxidant, but whether folic acid can be used to treat vitiligo is unclear. PIG1 cells that are an immortalized human epidermal melanocyte cell line were cultured in Medium 254. H2O2 at 800 μM was chosen to induce oxidative stress. CCK8 assay was used to estimate the cell survival rate. Flow cytometry was used to measure the intracellular ROS level and apoptosis rate. The nuclear translocation of Nrf2 was tested by immunofluorescence. Western blot and qRT-PCR were used to analyze the mRNA and protein expression levels of Keap1, Nrf2, HO-1, SOD2 and NQO1. We initially found that folic acid was able to ameliorate H2O2-induced oxidative damage in human melanocytes. In parallel, folic acid lessened the accumulation of intracellular ROS by potentiating the activity of antioxidant enzymes. Furthermore, folic acid protected melanocyte against oxidative stress by activating Nrf2 and its downstream genes HO-1, SOD2 and NQO1, and interfering with Nrf2 diminished the protection of folic acid against H2O2-induced apoptosis. At last, the down-regulation of Keap1 contributed to the activation of Nrf2 induced by folic acid.

158 Triggering immortalized keratinocyte growth arrest by protein kinase C delta activation

Normal epidermal keratinocyte (KC) differentiation is marked by irreversible cell cycle exit and a step-wise transition through the suprabasal layers of the epidermis. In contrast, KCs in both actinic keratosis and squamous cell carcinomas (SCCs) continue to proliferate in the suprabasal compartment, indicating a defect in their growth arrest signaling. Activation of multiple protein kinase C (PKC) isoforms have been associated with KC cell cycle withdrawal. To study the involvement of PKC in KC growth arrest, we evaluated the effects of the PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on normal human epidermal keratinocytes (NHEKs) and HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest over a 7 day period while untreated KCs underwent a 93-fold increase in cell number (p<0.005). 10 nM TPA treatment of HaCaT cells did not induce any growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells by flow cytometry. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment. In contrast, HaCaT-PKCδ-EGFP treated with TPA increased in cell number only 3-fold compared to a 15-fold increase in untreated HaCaT-PKCδ-EGFP over a 4 day growth assay. Activation of PKCδ in HaCaT-PKCδ-EGFP cells was confirmed by observing TPA-induced translocation of PKCδ-EGFP to the plasma membrane and increased S299 autophosphorylation. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics are being employed to identify PKCδ signaling pathways that can overcome the defective growth arrest in immortalized keratinocytes and may have therapeutic benefit for the treatment of actinic keratosis and SCCs.

Biotic / Abiotic Stress Influences on Human Epidermal Keratinocyte Cells

Human epidermal keratinocyte cells are the first defence blocks against the aggressive agents such as pathogens and xenobiotic materials. Synthesis process of the cellular proteins can be affected by abiotic stresses (aBS like: SiO2 nanoparticles) and biotic stresses (BS like: Virus), leading to the alteration of consumed energy profiles of proteins. The consumed energy profile of each protein was calculated based on their consumed ATP during the amino acids synthesizing procedure. Cells consumed more ATP, more energy, to synthesize more proteins under BS conditions compare to aBS conditions. Our results suggested that, the cells infected by pathogens are tend to survive longer than the treated cells by xenobiotic materials. Our data analysis revealed that the most energy reduction took place under aBS conditions. So, aBS could have severe effect on energy production pathways and decrease the energy source of cells. Moreover, the results demonstrated that the complexity of cellular protein networks under aBS conditions were more than BS conditions. It seems the cellular energy reduction under aBS conditions is one of the important factors in cell death. In addition, the position of proteins in the protein network was another important factor that should be carefully considered.

Solid-phase synthesis of molecularly imprinted polymer nanolabels: Affinity tools for cellular bioimaging of glycans

Hyaluronic acid (HA) is a glycosaminoglycan that plays many roles in health and disease and is a key biomarker of certain cancers. Therefore, its detection at an early stage, by histochemical methods, is of importance. However, intracellular HA can be masked by other HA-binding macromolecules, rendering its visualization somehow problematic. We show that fluorescent molecularly imprinted polymer nanogels (MIP-NPs), can localize and detect intracellular HA. MIP-NPs were synthesized by solid-phase synthesis on glass beads (GBs). GBs were functionalized with terminal alkyne groups on which an azide derivative of the template molecule glucuronic acid was immobilized via click chemistry. Immobilization via the anomeric carbon left the template’s carboxyl moiety free to enable strong stoichiometric electrostatic interactions with a benzamidine-based functional monomer, to confer selective recognition to the MIP-NPs. Due to the two-point orientation of the template, the resulting MIP-NPs were endowed with improved binding site homogeneity and specificity, reminiscent of monoclonal antibodies. These synthetic antibodies were then applied for probing and staining HA, of which glucuronic acid is a substructure (epitope), on human epidermal cells. Their excellent sensitivity, small size and water compatibility, enabled the MIP-NPs to visualize HA, as evidenced by confocal fluorescence micrographs.

Design, antimicrobial activity and mechanism of action of Arg-rich ultra-short cationic lipopeptides.

The increasing emergence of multidrug-resistant microorganisms represents one of the greatest challenges in the clinical management of infectious diseases, and requires the development of novel antimicrobial agents. To this aim, we de novo designed a library of Arg-rich ultra-short cationic antimicrobial lipopeptides (USCLs), based on the Arg-X-Trp-Arg-NH2 peptide moiety conjugated with a fatty acid, and investigated their antibacterial potential. USCLs exhibited an excellent antimicrobial activity against clinically pathogenic microorganisms, in particular Gram-positive bacteria, including multidrug resistant strains, with MIC values ranging between 1.56 and 6.25 μg/mL. The capability of the two most active molecules, Lau-RIWR-NH2 and Lau-RRIWRR-NH2, to interact with the bacterial membranes has been predicted by molecular dynamics and verified on liposomes by surface plasmon resonance. Both compounds inhibited the growth of S. aureus even at sub MIC concentrations and induced cell membranes permeabilization by producing visible cell surface alterations leading to a significant decrease in bacterial viability. Interestingly, no cytotoxic effects were evidenced for these lipopeptides up to 50–100 μg/mL in hemolysis assay, in human epidermal model and HaCaT cells, thus highlighting a good cell selectivity. These results, together with the simple composition of USCLs, make them promising lead compounds as new antimicrobials.

Research of Luteoloside in promoting proliferation and migration of human epidermal stem cells

Objective To investigate the effects of Luteoloside on the proliferation and migration of human epidermal stem cells (EpSCs) and the proliferation of epidermal cells in skin tissue in vitro. Methods EpSCs were isolated from human skin tissue by type Ⅳ collagenase digestion and collagen fast adherence. EpSCs were identified by immunofluorescence staining of CK19 and β1 integrin. The effects of Luteoloside (0 to 5.0 μmol/L) on the proliferation of EpSCs were measured by MTT assay. The effects of Luteoloside on EpSCs migration and wound healing were observed by scratch-wound model in vitro. The effects of Luteoloside on the thickness of epidermis in vitro were observed by HE staining. Results EpSCs formed a cobblestone-shaped cell monolayer when cultured on a collagen coated dish. Immunofluorescence staining showed that more than 90% cells were CK19 and β1 integrin positive. Luteoloside at 0.1 to 1.0 μmol/L could significantly promote the EpSCs proliferation; Luteoloside at 1.0 μmol/L could significantly enhance EpSCs migration and scratch healing. Culture of skin tissues with 1.0 μmol/L Luteoloside for 5 days could significantly increase the thickness of the epidermis. Conclusion Luteoloside can promote the proliferation and migration of EpSCs, promote the proliferation of epidermal tissue, and play an important role in skin self-renewal and injury repair. Key words: Cell proliferation; In vitro; Luteoloside; Epidermal stem cells; Migration

Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness

In human skin the junction between epidermis and dermis undulates, the width and depth of the undulations varying with age and disease. When primary human epidermal keratinocytes are seeded on collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on the tips not only expressed higher levels of the stem cell marker β1 integrin but also had elevated E-cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor MAL were higher at the base. AFM measurements established that the Young’s modulus of cells on the tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent on Rho kinase activity and intercellular adhesion. On flat substrates the Young’s modulus of calcium-dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase. Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces exerted at intercellular junctions in response to undulations in the epidermal-dermal interface. Statement of significanceIn human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned according to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the undulations and provide sufficient topographical information for stem cells to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base. The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in response to the slope of the undulations.

Asperyellone pretreatment protects HaCaT cells from UVB irradiation induced oxidative damages: Assessment under in vitro and in vivo conditions and at molecular level.

The present study explores the UVB protective role of Asperyellone (AY), a secondary metabolite of Aspergillus niger strain AN01. The in vitro UVB protective efficacy of AY was studied using the Human Epidermal keratinocytes cells (HaCaT) cell line. The results suggest the appreciable scavenging of UVB-induced reactive oxygen species in the AY-pretreated cells compared with UVB control. Experimental results on the antioxidant enzymes (Catalase, SOD, LPO, and GPx) profile, histochemical, and molecular analyses support the UVB protective effect of AY in HaCaT cells. Further, the in vivo UVB protective efficacy of AY was studied using animal models and compared with that of commercially available UVB protective agents. Physical, biochemical, and molecular analyses of skin samples emphasized the UVB protective role of AY. Thus, the important beneficial effects of AY have been explored in the present study.

Improving dermal delivery of hydrophilic macromolecules by biocompatible ionic liquid based on choline and malic acid

The aim of this work was to develop biocompatible ionic liquids (ILs) for enhancement of dermal delivery of hydrophilic macromolecules. The ILs were prepared from malic acid and choline, which are commonly used as food additives and generally regarded as safe. The choline malate IL (CM-IL) was formed via ionic interactions, which has been validated by Fourier transform infrared spectroscopy and nuclear magnetic resonance. Analysis by differential scanning calorimetry confirmed a melting point of −65.2 °C for CM-IL. In skin penetration study, CM-IL was proved to be able to deliver a model hydrophilic macromolecule dextran into deep skin. The amount of dextran delivered to epidermis and dermis by CM-IL is approximately two folds that of by dextran solution. Enhanced in vivo skin penetration by CM-IL was proved as well. The fluorescence of FITC-dextran could be observed permeating pervasively throughout the skin from 4 h to 24 h, while in the control groups it was mainly concentrated in stratum cornea (SC) and hair follicles. In addition, CM-IL did not shown any irritation to mice skin within 7 days of successive treatment and any toxicity to human epidermal cells (HEK-A) within 24 h. In conclusion, CM-IL could be potentially used as enhancers or vehicles for dermal delivery of hydrophilic macromolecules.

BLNCR is a long non-coding RNA adjacent to integrin beta-1 that is rapidly lost during epidermal progenitor cell differentiation

As our understanding of transcriptional regulation improves so does our appreciation of its complexity. Both coding and (long) non-coding RNAs provide cells with multiple levels of control and thereby flexibility to adapt gene expression to the environment. However, few long non-coding RNAs (lncRNAs) have been studied in human epidermal stem cells. Here, we characterized the expression of 26 lncRNAs in human epidermal keratinocytes, 7 of which we found to be dynamically expressed during differentiation. We performed in depth analysis of a lncRNA located proximal to the epidermal stem cell marker integrin beta-1 (ITGB1) and transcribed in the opposite direction. We dubbed this gene Beta1-adjacent long non-coding RNA, or BLNCR, and found that its expression is regulated by p63 and AP1 transcription factors. Furthermore, BLNCR expression is regulated downstream the integrin and EGF signaling pathways that are key to epidermal stem cell maintenance. Finally, we found that BLNCR expression is rapidly reduced upon induction of differentiation, preceding the down regulation of integrin beta-1 expression. These dynamics closely mirror the loss of proliferative and adhesion capacity of epidermal stem cells in colony formation assays. Together, these results suggest that loss of BLNCR expression marks the switch from a proliferative state towards terminal differentiation in human epidermal stem cells.