Human Organotypic Skin Models Research Articles

Characterization of TGF-β signaling in a human organotypic skin model reveals that loss of TGF-βRII induces invasive tissue growth.

Transforming growth factor-β (TGF-β) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-β signaling pathway, including the ligands TGF-β1, TGF-β2, and TGF-β3, the receptor TGF-βRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-β signaling, the loss of TGF-β1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-βRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-βRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-βRII. This study provides a framework for exploring TGF-β signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Dissecting the Functional Selectivity of TGFβ Signaling Pathway Components Using Genome Engineered Human Organotypic Skin Models

Transforming growth factor β (TGFβ) signaling is essential for cell growth and differentiation. Yet, the role of the individual TGFβ signaling components in human tissue homeostasis and transformation is still not completely understood. Here we applied a combined strategy of genetic engineering, a human organotypic 3D skin model, and global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGFβ signaling pathway. Our results show that loss of Smad4-dependent TGFβ signaling affected skin homeostasis through regulation of ECM expression and cell cycle control. In contrast, deletion of TGFβRII resulting in the combined loss of Smad4-dependent and independent TGFβ signaling induced invasive growth through activation of p38 and ERK signaling. The study provides a framework for exploration of signaling pathways with genetic engineering, human 3D tissue models, and with global proteomics and phosphoproteomics.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Effects of autologous platelet-rich plasma injections on facial skin rejuvenation.

Autologous serum platelet-rich plasma (PRP) has been used to rejuvenate wrinkled and aged skin for years; however, the molecular mechanism for the positive effects of PRP on the skin remains unclear. The present study aimed to clarify the potential molecular mechanisms for the role of PRP in wrinkled and aged skin rejuvenation, and provide evidence for future clinical applications. A total of 30 healthy females were recruited for PRP treatment and signed informed consent was obtained. A total of 3 autologous PRP injections were administered to each patient with 15-day intervals between injections. The effects of PRP injections were evaluated using the VISIA® Complexion Analysis System and skin computed tomography. A human organotypic skin model was established and treated with PBS or PRP before ultraviolet (UV)-B light (10 mJ/cm2) irradiation. The distribution of the epidermal structure and dermal fibers were analyzed by hematoxylin and eosin and Masson's trichome staining. Expression of matrix metalloproteinase-1 (MMP-1), tyrosinase, fibrillin and tropoelastin was detected by reverse transcription-quantitative PCR, western blotting and immunofluorescence. The present results showed that PRP treatment improved skin quality in the participants. In addition, the VISIA® results showed that wrinkles, texture and pores were decreased in the PRP groups compared with the PBS treatment. The in vitro study demonstrated that PRP treatment ameliorated photoaging by inhibiting UV-B-induced MMP-1 and tyrosinase upregulation, and by inducing fibrillin and tropoelastin expression that was downregulated by UV-B. Collectively, it was demonstrated that PRP treatment ameliorated skin photoaging through regulation of MMP-1, tyrosinase, fibrillin and tropoelastin expression.

Inflammation-induced tissue damage mimicking GvHD in human skin models as test-platform for immunotherapeutics_suppl

Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies.

Inflammation-induced tissue damage mimicking GvHD in human skin models as test-platform for immunotherapeutics.

With cellular products being on the front run there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells finally allowing to seed these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. Afterwards, we were able to induce inflammation-based tissue damage by pre-stimulated mismatched allogeneic lymphocytes and/or inflammatory cytokine containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. The effects could be prevented by the addition of immunosuppressants to the models. Consequently, these models would harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. This would also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells which would otherwise be limited to animal models. Thus, the current test platform developed with the limitation given that no professional APC are in place could highly reduce animal testing for investigation of novel immune therapies.

Chitosan‐Based Heparan Sulfate Mimetics Promote Epidermal Formation in a Human Organotypic Skin Model

AbstractBiomimetic materials that replicate biological functions have great promise for use as therapeutics in regenerative medicine applications. Heparan sulfate (HS) is the natural binding partner for growth factors enabling longer half‐lives and potentiation of their signaling. In this study a water soluble chitosan‐arginine is modified with sulfate moieties in order to mimic the structure of HS. Sulfated chitosan‐arginine with a degree of sulfation of 58% binds fibroblast growth factor 2 with higher affinity than HS. The sulfated chitosan‐arginine also promotes epithelial cell migration and supports the formation of an expanded epidermis in an organotypic skin model. Furthermore, sulfated chitosan‐arginine promotes the expression of the HS proteoglycan, perlecan, by both epithelial and fibroblast cells. Perlecan itself modulates the activity of mitogens and is essential for the formation of the epidermis. The synthesized sulfated Ch‐Arg derivatives mimic HS, support formation of the epidermis, and thus have the potential to assist in wound healing.

Repeated short climatic change affects the epidermal differentiation program and leads to matrix remodeling in a human organotypic skin model.

Human skin is subject to frequent changes in ambient temperature and humidity and needs to cope with these environmental modifications. To decipher the molecular response of human skin to repeated climatic change, a versatile model of skin equivalent subject to “hot–wet” (40°C, 80% relative humidity [RH]) or “cold–dry” (10°C, 40% RH) climatic stress repeated daily was used. To obtain an exhaustive view of the molecular mechanisms elicited by climatic change, large-scale gene expression DNA microarray analysis was performed and modulated function was determined by bioinformatic annotation. This analysis revealed several functions, including epidermal differentiation and extracellular matrix, impacted by repeated variations in climatic conditions. Some of these molecular changes were confirmed by histological examination and protein expression. Both treatments (hot–wet and cold–dry) reduced the expression of genes encoding collagens, laminin, and proteoglycans, suggesting a profound remodeling of the extracellular matrix. Strong induction of the entire family of late cornified envelope genes after cold–dry exposure, confirmed at protein level, was also observed. These changes correlated with an increase in epidermal differentiation markers such as corneodesmosin and a thickening of the stratum corneum, indicating possible implementation of defense mechanisms against dehydration. This study for the first time reveals the complex pattern of molecular response allowing adaption of human skin to repeated change in its climatic environment.

109 Short and repeated climatic changes affect the epidermal differentiation program and lead to matrix remodeling in a human organotypic skin model

109 Short and repeated climatic changes affect the epidermal differentiation program and lead to matrix remodeling in a human organotypic skin model

IL-31 regulates differentiation and filaggrin expression in human organotypic skin models

Atopic dermatitis (AD) is an inflammatory skin disease affecting 10% to 20% of children and 1% to 3% of adults in industrialized countries. Enhanced expression of IL-31 is detected in skin samples of patients with AD, but its physiological relevance is not known. We sought to determine the role of IL-31 in skin differentiation. We used human 3-dimensional organotypic skin models with either primary keratinocytes or HaCaT keratinocytes with inducible IL-31 receptor α to evaluate the effect of IL-31. The consequences were studied by using histology, the expression of markers analyzed by immunofluoresence and quantitative RT-PCR, and gene expression arrays. We observed that IL-31 interferes with keratinocyte differentiation. Gene expression analysis revealed a limited set of genes deregulated in response to IL-31, including IL20 and IL24. In HaCaT keratinocytes with inducible IL-31 receptor α, IL-31 inhibited proliferation upon induction of IL-31 receptor α by inducing cell cycle arrest. As in primary cells, IL-31-treated HaCaT cells elicited a differentiation defect in organotypic skin models, associated with reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the stratum basale, and poor development of the stratum granulosum. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. The highly induced proinflammatory cytokines IL-20 and IL-24 were responsible for part of the effect on FLG expression and thus for terminal differentiation. Our study suggests that IL-31 is an important regulator of keratinocyte differentiation and demonstrates a link between the presence of IL-31 in skin, as found in patients with AD, and filaggrin expression.

Gene silencing in a human organotypic skin model

Here we present a simple and highly reproducible method which allows the study of the effects of a single gene knockdown in an organotypic skin model. Human keratinocytes (KC) were transfected with backbone-modified short interfering RNAs (siRNAs) specific for vascular endothelial growth factor (VEGF) and matriptase-1. Twenty-four hours later the transfected cells were seeded onto fibroblast collagen suspensions and allowed to build up a multilayered epidermis by culture at the air/medium interface for 7 days. Protein expression of both targeted genes remained down-regulated by more than 80% up to 8 days after transfection. As expected, VEGF knockdown by siRNA did not alter epidermis formation in our organotypic skin model. By contrast ablation of matriptase-1 led to aberrant KC differentiation and impaired filaggrin processing and resulted in an epidermal phenotype closely resembling that of matriptase-1 deficient mouse skin. Our results suggest that siRNA-mediated gene silencing is highly efficient in an organotypic skin model and readily allows the assessment of the roles of individual genes during terminal KC differentiation.