Erratum: Publisher's Note: "Skin-on-a-chip models: General overview and future perspectives" [APL Bioengineering 5, 030901 (2021)].

[This corrects the article DOI: 10.1063/5.0046376.].

Skin-on-a-chip models: General overview and future perspectives.

Over the last few years, several advances have been made toward the development and production of in vitro human skin models for the analysis and testing of cosmetic and pharmaceutical products. However, these skin models are cultured under static conditions that make them unable to accurately represent normal human physiology. Recent interest has focused on the generation of in vitro 3D vascularized skin models with dynamic perfusion and microfluidic devices known as skin-on-a-chip. These platforms have been widely described in the literature as good candidates for tissue modeling, as they enable a more physiological transport of nutrients and permit a high-throughput and less expensive evaluation of drug candidates in terms of toxicity, efficacy, and delivery. In this Perspective, recent advances in these novel platforms for the generation of human skin models under dynamic conditions for in vitro testing are reported. Advances in vascularized human skin equivalents (HSEs), transferred skin-on-a-chip (introduction of a skin biopsy or a HSE in the chip), and in situ skin-on-a-chip (generation of the skin model directly in the chip) are critically reviewed, and currently used methods for the introduction of skin cells in the microfluidic chips are discussed. An outlook on current applications and future directions in this field of research are also presented.

A new microfluidic method enabling the generation of multi-layered tissues-on-chips using skin cells as a proof of concept.

Microfluidic-based tissues-on-chips (TOCs) have thus far been restricted to modelling simple epithelia as a single cell layer, but likely due to technical difficulties, no TOCs have been reported to include both an epithelial and a stromal component despite the biological importance of the stroma for the structure and function of human tissues. We present, for the first time, a novel approach to generate 3D multilayer tissue models in microfluidic platforms. As a proof of concept, we modelled skin, including a dermal and an epidermal compartment. To accomplish this, we developed a parallel flow method enabling the deposition of bilayer tissue in the upper chamber, which was subsequently maintained under dynamic nutrient flow conditions through the lower chamber, mimicking the function of a blood vessel. We also designed and built an inexpensive, easy-to-implement, versatile, and robust vinyl-based device that overcomes some of the drawbacks present in PDMS-based chips. Preliminary tests indicate that this biochip will allow the development and maintenance of multilayer tissues, which opens the possibility of better modelling of the complex cell-cell and cell-matrix interactions that exist in and between the epithelium and mesenchyme, allowing for better-grounded tissue modelling and drug screening.

An inactivating CYLD mutation promotes skin tumor progression by conferring enhanced proliferative, survival and angiogenic properties to epidermal cancer cells.

In this study, we demonstrate that the expression in tumorigenic epidermal cells of a catalytically inactive form of CYLD (CYLD(C/S)) that mimics the identified mutations of cyld in human tumors and competes with the endogenous CYLD results in enhanced cell proliferation and inhibition of apoptosis; it also stimulates cell migration and induces the expression of angiogenic factors, including vascular endothelial growth factor-A. Altogether, these characteristics indicate an increased oncogenicity of the tumorigenic epidermal CYLD(C/S) mutant cells in vitro. Moreover, we show the increase in malignancy of epidermal squamous cell carcinomas that express the CYLD(C/S) transgene in an in vivo xenograft model. Tumors carrying the mutated CYLD(C/S) exhibit a fast growth, are poorly differentiated and present a robust angiogenesis. CYLD(C/S) tumors are also characterized by their elevated proliferation rate and decreased apoptosis. In contrast with previous studies showing the development of benign tumors by mutations in the CYLD gene, here we provide evidence that the occurrence of mutations in the CYLD gene in tumorigenic epidermal cells (carrying previous mutations) increases the aggressiveness of carcinomas, mainly through enhancement of the expression of angiogenic factors, having therefore a key role in epidermal cancer malignancy.

The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. OBJECTIVES: To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype-phenotype correlations. METHODS: Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. RESULTS: Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46.3% of Spanish RDEB alleles. A consistent genotype-phenotype correlation was established. CONCLUSIONS: Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

Skin gene therapy for acquired and inherited disorders.

The rapid advances associated with the Human Genome Project combined with the development of proteomics technology set the bases to face the challenge of human gene therapy. Different strategies must be evaluated based on the genetic defect to be corrected. Therefore, the re-expression of the normal counterpart should be sufficient to reverse phenotype in single-gene inherited disorders. A growing number of candidate diseases are being evaluated since the ADA deficiency was selected for the first approved human gene therapy trial (Blaese et al., 1995). To cite some of them: sickle cell anemia, hemophilia, inherited immune deficiencies, hyper-cholesterolemia and cystic fibrosis. The approach does not seem to be so straightforward when a polygenic disorder is going to be treated. Many human traits like diabetes, hypertension, inflammatory diseases and cancer, appear to be due to the combined action of several genes and environment. For instance, several wizard gene therapy strategies have recently been proposed for cancer treatment, including the stimulation of the immune system of the patient (Xue et al., 2005), the targeting of particular signalling pathways to selectively kill cancer cells (Westphal and Melchner, 2002) and the modulation of the interactions with the stroma and the vasculature (Liotta, 2001; Liotta and Kohn, 2001).

Conditional ablation of C/EBP beta demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis.

The CCAAT/enhancer binding protein beta (C/EBP beta) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBP beta (C/EBP beta(-/-)) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBP beta(-/-) mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBP beta to specific phenotypes, mice with a conditional 'floxed' C/EBP beta null allele were generated. Epidermal-specific deletion of C/EBP beta was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBP beta(-/-) mice, K5-Cre;C/EBP beta(fl/fl) mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBP delta, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBP beta in skin tumor development. Our findings demonstrate that C/EBP beta exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBP beta in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.

Hair cycle and wound healing in mice with a keratinocyte-restricted deletion of FAK.

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

Current approaches and perspectives in human keratinocyte-based gene therapies.

Inherited and acquired disorders are liable to treatment with somatic gene therapy. The skin, and in particular epidermal cells, are particularly suited to genetic manipulation and follow-up of therapeutic effects. Cutaneous gene therapy may be effective for skin defects and systemic abnormalities. The robust basic and preclinical data available today would support the application of keratinocyte-based gene therapy to patients.

Abnormal cochlea linked to deafness in transgenic mice expressing human cytokeratin K8.

The cytokeratin intermediate filaments have a relevant role in the proliferation and differentiation processes of epithelial cells. To provide information about the role of K8 cytokeratin during the auditory receptor differentiation, two groups of adult mice were used: TGK8-4 transgenic and control animals. The TGK8-4 transgenic mice contained 12 kb of K8 human cytokeratin (HK8) locus (Casanova et al., 1995, 1999). The functional activity of the auditory receptor was analyzed by auditory thresholds. Morphological studies demonstrate that the auditory receptors of the TGK8-4 transgenic mice are highly immature. Immunocytochemical studies were made by using two monoclonal antibodies: CAM 5-2 (recognizing K8 human cytokeratin) and Troma-1 (recognizing both mouse and human K8 cytokeratin). These demonstrated significant differences between the auditory receptors of the transgenic mice and the control mice. These functional and morphological differences clearly suggest that K8 cytokeratin has a relevant role during the differentiation and tridimensional organization of the sensory and the supporting cells of the auditory receptor.

Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis.

Differentiation-related gene-1 (Drg-1) has been identified as a gene whose expression is increased in several processes related to differentiation, but its function is currently unknown. In this report, we show that Drg-1 was expressed in keratinocytes, this expression being rapidly increased as a result of induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the presence of an activating form of Ha-ras. Induction by TPA occurred both in cultured cell lines and primary keratinocytes as well as in mouse skin after a single TPA application. Overexpression of Drg-1 was also observed in TPA-induced hyperplastic skin. In agreement, mouse skin papillomas and carcinomas also overexpressed Drg-1. In addition, Drg-1 was induced when keratinocytes were forced to differentiate by calcium switch or serum starvation. Analysis of the expression of Drg-1 during the keratinocyte cell cycle demonstrated relatively high levels of Drg-1 mRNA in G(0), which increased in early G(1) and decreased afterwards in late G(1)/S. In situ analysis showed an accumulation of Drg-1 in the suprabasal layers of the skin, as well as in the more differentiated areas of mouse skin papillomas. These results suggest that, in addition to being upregulated during keratinocyte differentiation, the Drg-1 gene might have a complex function in skin tumorigenesis.

Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest.

The intermediate filament cytoskeleton is composed of keratins in all epithelial cells and imparts mechanical integrity to these cells. However, beyond this shared function, the functional significance of the carefully regulated tissue- and differentiation-specific expression of the large keratin family of cytoskeletal proteins remains unclear. We recently demonstrated that expression of keratin K10 or K16 may regulate the phosphorylation of the retinoblastoma protein (pRb), inhibiting (K10) or stimulating (K16) cell proliferation (J. M. Paramio, M. L. Casanova, C. Segrelles, S. Mittnacht, E. B. Lane, and J. L. Jorcano, Mol. Cell. Biol. 19:3086-3094, 1999). Here we show that keratin K10 function as a negative modulator of cell cycle progression involves changes in the phosphoinositide 3-kinase (PI-3K) signal transduction pathway. Physical interaction of K10 with Akt (protein kinase B [PKB]) and atypical PKCzeta causes sequestration of these kinases within the cytoskeleton and inhibits their intracellular translocation. As a consequence, the expression of K10 impairs the activation of PKB and PKCzeta. We also demonstrate that this inhibition impedes pRb phosphorylation and reduces the expression of cyclins D1 and E. Functional and biochemical data also demonstrate that the interaction between K10 and these kinases involves the non-alpha-helical amino domain of K10 (NTerm). Together, these results suggest new and essential roles for the keratins as modulators of specific signal transduction pathways.

The ink4a/arf tumor suppressors cooperate with p21cip1/waf in the processes of mouse epidermal differentiation, senescence, and carcinogenesis.

In mammalian cells, cell cycle withdrawal is a prerequisite for terminal differentiation. Accordingly, in most tissues, including epidermis, the expression of the cyclin-dependent kinase inhibitors increases during differentiation. However, the actual role of cyclin-dependent kinase inhibitors is unclear. Different aspects of epidermal growth and differentiation in ink4a(Delta2,3)-null, p21-null, and ink4a(Delta2,3)/p21-doubly deficient mice were studied. Altered differentiation and decreased age-related senescence were found in the epidermis of ink4a(Delta2,3)/p21-null mice and, to a lesser extent, in ink4a(Delta2,3)- and p21-null mice. ink4a(Delta2,3)/p21-null primary keratinocytes underwent cell cycle arrest upon calcium or transforming growth factor-beta treatment, but failed to differentiate. This differentiation deficiency was not observed in p21- or ink4a(Delta2,3)-deficient keratinocytes. Upon infection with a v-Ha-ras-coding retrovirus, wild-type keratinocytes displayed features indicative of premature cell senescence. In p21- or ink4a(Delta2,3)-deficient keratinocytes, only a partial response was observed. ink4a(Delta2,3)/p21-deficient keratinocytes did not display senescent features, but showed increased tumorigenic potential upon injection into nude mice. These results indicate that ink4a/arf and cip1/waf genes cooperate to allow normal keratinocyte differentiation and that the absence of both favors malignant transformation.

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor.

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene-transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin-producing HK were transplanted to immunosuppressed ob/ob mice. HK-derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age-associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase-transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild-type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase-transgenic mice also shows an increased wound-healing rate compared with wild-type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age-associated diseases based on telomerase upregulation.

Opposite functions for E2F1 and E2F4 in human epidermal keratinocyte differentiation.

Proteins of the retinoblastoma family (pRb, p107, and p130) modulate cell proliferation, a function related to their capacity to control the activity of the E2F transcription factor family. The Rb proteins also control cell differentiation in different tissues. We have recently described their involvement in human epidermal keratinocyte differentiation (Paramio, J. M., Lain, S., Segrelles, C., Lane, E. B. , and Jorcano, J. L. (1998) Oncogene 17, 949-957). Here we show that E2F proteins are also involved in this process. We found that E2F1 and E2F4 are expressed differentially during the in vitro differentiation of human epidermal keratinocytes, with the former uniformly present throughout the process, whereas the second is predominantly expressed at the onset of differentiation. This pattern is also observed in human skin by confocal microscopy. Electrophoretic mobility shift assays and immunoprecipitation experiments demonstrated that the complexes formed by E2F1 and E2F4 and Rb family proteins vary throughout in vitro keratinocyte differentiation. In agreement with this observation, several E2F-responsive genes are differentially regulated during this process. To test the functional implications of these observations, we transfected HaCaT keratinocytes with plasmids coding for E2F1 and E2F4. Transfected cells display opposite in vitro differentiation properties. Although E2F1-transfected cells are unable to differentiate, E2F4-transfected cells show an increased differentiation rate compared with Neo-transfected control cells. Our data demonstrate that the differential and coordinated expression and interaction of E2F and Rb proteins modulate the process of epidermal differentiation and provide clear evidence that members of the E2F family of transcription factors play specific and opposite roles during cell differentiation.

Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia.

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.

Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes.

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.