3D Human Skin Equivalents Research Articles

228 Loss of EPHA2 represses GATA-3 function and causes a terminal differentiation defect

Epidermal morphogenesis and differentiation require the coordination of complex signal transduction networks. These signal relays are often initiated at the plasma membrane and transmitted to the nucleus to control gene expression. Receptor tyrosine kinases (RTKs) are integral in orchestrating communication cascades to induce differentiation. We report loss of EPHA2 RTK causes a terminal differentiation defect in 3D human skin equivalents (3D HSE) resulting in loss of stratification and ablation of the granular and cornified layers. In EPHA2-deficient (shEPHA2) 3D HSE, we show significant loss (P<0.05) of loricrin, filaggrin, and involucrin protein and mRNA, indicating EPHA2 signaling can impact keratinocyte differentiation at the transcriptional level. The transcription factor GATA-3 is a key driver of differentiation-associated gene expression in epidermis, although the upstream signals regulating its activity are unknown. GATA-3 is expressed in the nuclei of suprabasal keratinocytes in mature 3D HSE, mimicking the pattern in normal human skin (R2=0.99). In 2D cultures, GATA-3 accumulates in the nucleus following 24 h exposure to 1.2 mM calcium. However, GATA-3 protein and transcript expression are lost in shEPHA2 3D HSE and 2D cultures (P<0.001). Further, there is a decrease of GATA-driven transcription indicated by a significant loss in GATA binding activity in a promoter luciferase reporter assay (P<0.001). Re-expression of nuclear GATA-3 using a retroviral construct in EPHA2-deficient 3D HSE restored differentiation. Taken together, these results indicate that EPHA2 promotes GATA-3 nuclear accumulation to positively regulate the transcription of terminal differentiation genes in epidermal keratinocytes.

564 Melasolv, a skin lightening compound with different mechanisms to regulate skin pigmentation

The pigmentation of human skin (nevi, senile lentigines, melasma, etc) is regulated by a complex process involving the synthesis and distribution of melanin. Overexpression of melanin is induced by the off-balance between the signals which regulates melanin synthesis, this can be caused due to a response to external or internal stimulus that often affects the genes related to melanogenesis. To date, many studies have focused on developing direct enzymatic inhibitors of tyrosinase in order to achieve a skin-lightening effect. However, it was also reported that these direct inhibitors could be converted to quinone derivatives by tyrosinase-catalyzed oxidation, which lead to undesirable outcomes. Therefore, we have studied intrinsic inhibitory factors of melanogenesis in skin with the objective to achieve a lightening effect without the undesirable potential side effects. It was reported that non-coding RNAs including H19, miR125b and WIF1 were decreased in hyper-pigmented skin such as melasma and nevi. And it was found out that melanogenesis could be inhibited by increasing these lightening genes expression in normal human epidermal melanocytes and keratinocytes. Our group showed that the 3, 4, 5-trimethoxy cinnamate thymol ester (TCTE, Melasolv®), synthesized from gallic acid, is a lightening active which decreases tyrosinase protein formation by regulating the MITF. Likewise, we have also observed that Melasolv significantly decreased melanogenesis by increasing expression of the lightening genes H19, miR125b and WIF1 in the skin cells. Finally, we have confirmed the lightening effect of Melasolv in 3D human skin equivalent. Nonetheless, since Melasolv is not a direct inhibitor of tyrosinase enzymatic activity, it is extremely unlikely that nocive quinone derivatives conversions occur. From these results, we have proposed Melasolv as an active for lightening effects with a safer profile, when considering undesirable effects, to treat pigmentary disorder and uneven skin color.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

Small Molecule IL-36\u03b3 Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.

361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.

Abstract LB-355: Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes

Abstract Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. We previously demonstrated that UV radiation reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1), and that loss-of-function mutations in Dsg1 increased keratinocyte cytokine production in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. Based on these observations we hypothesized that loss of Dsg1 alters the microenvironment through increased cytokine signaling. Here, we show that Dsg1 expression is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas. In contrast, keratinocyte E-cadherin was not significantly altered in early tumors. Dsg1 knockdown in keratinocytes in vitro increased phospho-EGFR, phospho-Stat3, and nuclear NFκB, while increasing keratinocyte mRNAs encoding IL-1α, IL-1β, IL-6 and IL-8 pro-inflammatory cytokines. Re-expressing silencing-resistant Dsg1 returned cytokine levels to control levels. To determine whether Dsg1 loss in keratinocytes elicits paracrine-mediated changes in melanocyte behavior, we treated normal primary melanocytes with conditioned media from Dsg1-silenced keratinocytes. Short-term (overnight) exposure to Dsg1-deficient conditioned media resulted in significantly increased melanocyte dendrite length, similar to morphology changes induced by UV exposure; however, long-term (7 day) exposure resulted in significantly shorter dendrites. Melanocytes exposed to Dsg1-deficient media increased melanin secretion and decreased melanin retention, a phenomenon associated with the process of transformation. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 functions to maintain baseline levels of cytokine signaling, but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation. Citation Format: Christopher Arnette, Jodi L. Johnson, Hope E. Burks, Jennifer L. Koetsier, Kathleen J. Green. Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-355.

Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy.

Three-dimensional skin equivalents are increasingly gaining acceptance as non-animal based experimental models of human skin. They are particularly suited to studying differences in physical and compositional properties of normal and diseased skin and their impact on the skin's barrier function. Typically, a culture protocol yielding a model of normal skin is modified to create a model simulating a pathology. Skin layer thicknesses and lipid/protein contents are compared using methods that are invasive, precluding further experiments on the same replicates, and which may be prone to artefacts. We show here that confocal Raman spectroscopy (CRS) is a valuable method for non-invasive discrimination of skin equivalents grown under different culture conditions. Using 3D full-thickness skin equivalents developed in-house, we measure significant differences in stratum corneum and viable epidermis apparent thicknesses resulting from a 7-day difference in the cultures' air-lift phase and from supplementation of the culture medium with interleukin 4. Furthermore, stratum corneum thicknesses obtained by CRS are up to 2.6-fold higher than values measured from histological photomicrographs. Regarding composition, CRS reveals the differential effects of the culture protocol modifications on ceramide, cholesterol and protein composition as a function of depth in the stratum corneum.

Correction: Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy.

Correction for 'Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy' by Y. Dancik, et al., Analyst, 2018, DOI: .

Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target

Skin fibrosis in systemic sclerosis (SSc) is accompanied by attrition of dermal white adipose tissue (dWAT) and reduced levels of circulating adiponectin. Since adiponectin has potent regulatory effects on fibroblasts, we sought to assess adiponectin signaling in SSc skin biopsies, and evaluate fibrosis in mice with adiponectin gain- and loss-of-function mutations. Furthermore, we investigated the effects and mechanism of action of agonist peptides targeting adiponectin receptors in vitro and in vivo. We found that adiponectin pathway activity was significantly reduced in a subset of SSc skin biopsies. Mice lacking adiponectin mounted an exaggerated dermal fibrotic response, while transgenic mice with constitutively elevated adiponectin showed selective dWAT expansion and protection from skin and peritoneal fibrosis. Adiponectin receptor agonists abrogated ex vivo fibrotic responses in explanted normal and SSc fibroblasts and in 3D human skin equivalents, in part by attenuating focal adhesion complex assembly, and prevented and reversed experimentally-induced organ fibrosis in mice. These results implicate aberrant adiponectin pathway activity in skin fibrosis, identifying a novel function for this pleiotropic adipokine in regulation of tissue remodeling. Restoring adiponectin signaling in SSc patients therefore might represent an innovative pharmacological strategy for intractable organ fibrosis.

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70.

806 Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes

Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. Supporting this idea, we observed reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1) (which we previously showed is lost in response to UV) in regions surrounding human in situ and stage I/II melanomas. Loss of Dsg1 was specific as expression of E-cadherin was not significantly altered in early tumors. Dsg1-deficient keratinocytes also exhibited indications of impaired Dsg1-dependent signaling including increased phospho-EGFR and nuclear NFκB. To directly determine whether Dsg1 loss affects melanocyte behavior, we carried out in vitro analyses of Dsg1-silenced keratinocytes. Dsg1 knockdown increased keratinocyte expression of mRNAs encoding multiple pro-inflammatory cytokines, which also occurred in primary human melanocytes co-cultured with these keratinocytes. Growth of melanocytes in media conditioned by Dsg1-deficient keratinocytes resulted in significantly increased average melanocyte dendrite length (104μm vs. 74μm in controls; p=0.002) and dendrite number (3.3 vs. 2.6/cell; p=0.001). Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 keeps cytokine signaling in check but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation and/or progression. We propose Dsg1 loss as a potential biomarker for increased melanoma risk and a therapeutic target for histone deacetylase inhibitors, which we have shown increase Dsg1 expression.

The "Alarmins" HMBG1 and IL-33 Downregulate Structural Skin Barrier Proteins and Impair Epidermal Growth.

The epidermal-derived "alarmins" high-mobility group box 1 (HMGB1) protein and interleukin-33 (IL-33) are upregulated in patients with atopic dermatitis. How-ever, their capacity as pro-inflammatory cytokines and their derived effects on skin barrier regulation are poorly elucidated. We investigated the impact of HMGB1 and IL-33 on gene transcription, protein expression and epidermal differentiation across 3 distinct keratinocyte in vitro models. Primary keratinocytes from healthy donors were used in submerged monolayer cultures, 3D human epidermis equivalents and 3D human skin equivalents. All keratinocyte models underwent 96-h stimulation with HMGB1 (100 μM) or IL-33 (100 ng/ml) using IL-4 (50 ng/ml) as positive control of regulation and vehicle as negative control. We found that HMGB1 and IL-33 downregulated transcription of several genes from members of the epidermal differentiation complex, including filaggrin. Furthermore, HMGB1 downregulated the expression of the encoded proteins in the upper epidermis. Finally, IL-33 and HMGB1 ultimately led to impaired epidermal growth and maturation. In conclusion, HMGB1 and IL-33 could play a significant role in the atopic dermatitis pathophysiology due to negative regulation of structural proteins, stratum corneum formation and epidermal growth.

The Lipophilic Vitamin C Derivative, 6-O-Palmitoylascorbate Protects Human Keratinocytes and 3D-Human Skin Equivalents Against X-Ray-Induced Oxidative Stress and Apoptosis More Markedly Than L-Ascorbic Acid.

The aim of this study was to investigate preventive effects of the lipophilic vitamin C derivative, 6-O-palmitoylascorbate (PlmtVC) against X-ray radiation-induced harmful events. Free radical scavenging activity tests showed that both fresh and old (being kept at 37°C for 72 h) solutions of PlmtVC showed significantly higher abilities for scavenging both DPPH and peroxyl radical (ROO·) radicals than L-ascorbic acid (L-AA) under the same conditions, suggesting that PlmtVC is an antioxidant more efficient and stable than L-AA. Irradiation with X-ray (15 Gy) increased intracellular ROS production, lipid peroxidation and protein carbonylation, in human keratinocytes HaCaT, all of which were repressed, especially for intracellular ROS more markedly, by PlmtVC than by L-AA. After X-ray (15 Gy)-irradiation, caspase 3/7 activation and TUNEL-detected DNA-strand-breakages characteristic of apoptosis obviously increased in HaCaT cells or 3D-skin tissue equivalents, respectively, both of which were prevented more appreciably by PlmtVC than by L-AA. PlmtVC also noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-skin equivalents. Thus, PlmtVC prevents X-ray-induced diverse harmful effects, through its antioxidant activity and the palmitoyl moiety-based lipophilicity, more efficiently than L-AA. J. Cell. Biochem. 118: 318-329, 2017. © 2016 Wiley Periodicals, Inc.

Human Skin Constructs with Spatially Controlled Vasculature Using Primary and iPSC-Derived Endothelial Cells.

Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival, functionality, and viable engraftment. In this study, the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function, which allows for the recapitulation of both topical and systemic delivery of drugs. In addition, the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall, this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds.

In vitro methods for screening transdermal formulations.

This article provides a review of the critical in vitro assays utilized in transdermal drug development. In vitro assays such as percutaneous absorption testing and dissolution (drug release) testing are powerful tools for screening potential transdermal compounds and drug quality control, respectively. Several 2D single-cell cultures and 3D human skin equivalents are available for screening compounds with low irritation and sensitization potential. The role of each assay and its limitations and challenges will be further discussed below.

Evaluation of 3D-human skin equivalents for assessment of human dermal absorption of some brominated flame retardants

Ethical and technical difficulties inherent to studies in human tissues are impeding assessment of the dermal bioavailability of brominated flame retardants (BFRs). This is further complicated by increasing restrictions on the use of animals in toxicity testing, and the uncertainties associated with extrapolating data from animal studies to humans due to inter-species variations. To overcome these difficulties, we evaluate 3D-human skin equivalents (3D-HSE) as a novel in vitro alternative to human and animal testing for assessment of dermal absorption of BFRs. The percutaneous penetration of hexabromocyclododecanes (HBCD) and tetrabromobisphenol-A (TBBP-A) through two commercially available 3D-HSE models was studied and compared to data obtained for human ex vivo skin according to a standard protocol. No statistically significant differences were observed between the results obtained using 3D-HSE and human ex vivo skin at two exposure levels. The absorbed dose was low (less than 7%) and was significantly correlated with log Kow of the tested BFR. Permeability coefficient values showed increasing dermal resistance to the penetration of γ-HBCD>β-HBCD>α-HBCD>TBBPA. The estimated long lag times (>30min) suggests that frequent hand washing may reduce human exposure to HBCDs and TBBPA via dermal contact.

Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.

Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10μM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.

A pilot study of the photoprotective effect of almond phytochemicals in a 3D human skin equivalent

UV exposure causes oxidative stress, inflammation, erythema, and skin cancer. α-Tocopherol (AT) and polyphenols (AP) present in almonds may serve as photoprotectants. Our objectives were to assess the feasibility of using a 3D human skin equivalent (HSE) in photoprotectant research and to determine photoprotection of AT and AP against UVA radiation. AT or AP was applied to medium (25 and 5μmol/L, respectively) or topically (1mg/cm2 and 14μg/cm2), followed by UVA. Photodamage assessed 96h post UVA included HSE morphology, keratinocyte proliferation, apoptosis, and differentiation. UVA induced disorganization of basal layer, alteration of epidermal development, and fibroblast loss which were alleviated by all nutrient pretreatments. UVA significantly decreased keratinocyte proliferation compared to controls, and all pretreatments tended to negate the reduction though only the medium AT effect was statistically significant (p⩽0.05). UVA led to a significant 16-fold increase in apoptosis of fibroblasts compared to the control which was alleviated by topical AP pretreatment and completely negated by topical AT (p⩽0.05). In conclusion, we validated the feasibility of using HSE in evaluation of photoprotectants and found that AT and AP, applied to medium or topically, provided some degree of photoprotection against UVA.

Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents

IntroductionPluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation.MethodsIn this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs.ResultsWhile both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF.ConclusionsOur findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation.

The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents

Epithelial–mesenchymal interactions promote the morphogenesis and homeostasis of human skin. However, the role of the basement membrane (BM) during this process is not well-understood. To directly study how BM proteins influence epidermal differentiation, survival and growth, we developed novel 3D human skin equivalents (HSEs). These tissues were generated by growing keratinocytes at an air–liquid interface on polycarbonate membranes coated with individual matrix proteins (Type I Collagen, Type IV Collagen or fibronectin) that were placed on contracted Type I Collagen gels populated with dermal fibroblasts. We found that only keratinocytes grown on membranes coated with the BM protein Type IV Collagen showed optimal tissue architecture that was similar to control tissues grown on de-epidermalized dermis (AlloDerm) that contained intact BM. In contrast, tissues grown on proteins not found in BM, such as fibronectin and Type I Collagen, demonstrated aberrant tissue architecture that was linked to a significant elevation in apoptosis and lower levels of proliferation of basal keratinocytes. While all tissues demonstrated a normalized, linear pattern of deposition of laminin 5, tissues grown on Type IV Collagen showed elevated expression of α6 integrin, Type IV Collagen and Type VII Collagen, suggesting induction of BM organization. Keratinocyte differentiation (Keratin 1 and filaggrin) was not dependent on the presence of BM proteins. Thus, Type IV Collagen acts as a critical microenvironmental factor in the BM that is needed to sustain keratinocyte growth and survival and to optimize epithelial architecture.