Efficient Generation of CRISPR/Cas9-Mediated Knockout Human Primary Keratinocytes by Electroporation.

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.

The Endogenous Dual Retinoid Receptor Agonist Alitretinoin Exhibits Immunoregulatory Functions on Antigen-Presenting Cells.

Retinoids are a frequently used class of drugs in the treatment of inflammatory as well as malignant skin diseases. Retinoids have differential affinity for the retinoic acid receptor (RAR) and/or the retinoid X receptor (RXR). The endogenous dual RAR and RXR agonist alitretinoin (9-cis retinoic acid) demonstrated remarkable efficacy in the treatment of chronic hand eczema (CHE) patients; however, detailed information on the mechanisms of action remains elusive. Here, we used CHE as a model disease to unravel immunomodulatory pathways following retinoid receptor signaling. Transcriptome analyses of skin specimens from alitretinoin-responder CHE patients identified 231 significantly regulated genes. Bioinformatic analyses indicated keratinocytes as well as antigen presenting cells as cellular targets of alitretinoin. In keratinocytes, alitretinoin interfered with inflammation-associated barrier gene dysregulation as well as antimicrobial peptide induction while markedly inducing hyaluronan synthases without affecting hyaluronidase expression. In monocyte-derived dendritic cells, alitretinoin induced distinct morphological and phenotypic characteristics with low co-stimulatory molecule expression (CD80 and CD86), the increased secretion of IL-10 and the upregulation of the ecto-5'-nucleotidase CD73 mimicking immunomodulatory or tolerogenic dendritic cells. Indeed, alitretinoin-treated dendritic cells demonstrated a significantly reduced capacity to activate T cells in mixed leukocyte reactions. In a direct comparison, alitretinoin-mediated effects were significantly stronger than those observed for the RAR agonist acitretin. Moreover, longitudinal monitoring of alitretinoin-responder CHE patients could confirm in vitro findings. Taken together, we demonstrate that the dual RAR and RXR agonist alitretinoin targets epidermal dysregulation and demonstrates strong immunomodulatory effects on antigen presenting cell functions.

Platelet lysate can support the development of a 3D-engineered skin for clinical application.

Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90+FAP+, CD90+FAP- and CD90-FAP+, based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90+ FAP+ subpopulation in expense of reticular CD90+FAP-, which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-ß1 was identified as one of the most abundant. TGF-ß1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-ß1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections.

BACKGROUND: Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination. OBJECTIVES: We sought to identify circulatory and skin lipid biomarkers associated with EH and EV. METHODS: Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication in vitro. RESULTS: The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P < .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P < .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean = 1,533 vs 732 pmol/mL; P < .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes. CONCLUSIONS: Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Platelet lysate promotes the healing of long-standing diabetic foot ulcers: A report of two cases and in vitro study.

Long-standing foot ulcers present a great challenge in diabetes care. Platelet products have been suggested as a possible therapeutic option. However, nor the effect of an injectable form of platelet lysate on the healing of ulcers nor that on primary cells of the epidermis have been studied. In the current study, we present two cases of an ongoing clinical trial showing the positive effect of autologous platelet lysate injected perilesional. Both clinical cases treated with injections of hPL showed complete healing of previously un-healed within 8 weeks of treatment. Further, we describe the in vitro effect of human platelet lysate (hPL) on primary human epidermal keratinocytes (HEK) in terms of chemotaxis, migration and proliferation. In vitro, HEK showed enhanced chemotaxis towards the hPL compared to keratinocyte-defined media (p < 0.0001). Their migration was also stimulated especially at hPL concentration of 10%V/V (p < 0.0001). In contrast, hPL significantly inhibited HEK proliferation measured through MTT assay (p < 0.0001). In conclusion, the findings presented here provide preliminary evidence of an explanatory mechanism for the effect of hPL on primary keratinocytes and therefore of their potential use in a clinical setting. hPL promotes keratinocyte migration and therefore closure of foot ulcers.

Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.

The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.

Down-Regulation of MHC Class I Expression in Human Keratinocytes Using Viral Vectors Containing US11 Gene of Human Cytomegalovirus and Cultivation on Bovine Collagen-Elastin Matrix (Matriderm®): Potential Approach for an Immune-Privileged Skin Substitute.

Skin transplantation, especially in burn patients, is still challenging because surgeons are faced with limited disposability of autologous donor side material. The in vitro culture of keratinocytes has become an important reconstructive option. However, only non-immunogenic allogenic keratinocytes offer the opportunity to develop a skin graft that can overcome rejection. The purpose of the study was to develop targeted gene modification of keratinocytes in order to reduce immunogenicity for the use as allogenic transplantable skin graft by decreasing the expression of MHC class I. To reduce MHC class I expression, viral vectors containing the US11 gene of human cytomegalovirus were generated and tested on their functionality using Western blotting, indirect immunofluorescence staining, and flow cytometry. Transfected keratinocytes were seeded on commercially available bovine collagen-elastin matrices and further cultured for histological and cell survival assays. Results showed transient down-regulation of MHC class I after 24 h post-transfection, with recovery of MHC class I expression after 48 h. Histological assessments showed long-term cell survival as well as histological patterns comparable to epidermal layers of healthy human skin. The data postulates the potential application of US11 transfected keratinocytes as an approach towards an immune-privileged skin substitute. Nevertheless, further studies and data are needed.

Conditional ablation of p130Cas/BCAR1 adaptor protein impairs epidermal homeostasis by altering cell adhesion and differentiation.

BACKGROUND: p130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis. METHODS: To this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue. RESULTS: By using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of "late" epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population. CONCLUSION: Altogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.

Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair.

Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.

Functional Analysis of LINC Complexes in the Skin.

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.

Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.

BACKGROUND: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. METHODS: Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. RESULTS: Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. CONCLUSIONS: Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.

In Vitro Dermal Safety Assessment of Silver Nanowires after Acute Exposure: Tissue vs. Cell Models.

Silver nanowires (AgNW) are attractive materials that are anticipated to be incorporated into numerous consumer products such as textiles, touchscreen display, and medical devices that could be in direct contact with skin. There are very few studies on the cellular toxicity of AgNW and no studies that have specifically evaluated the potential toxicity from dermal exposure. To address this question, we investigated the dermal toxicity after acute exposure of polymer-coated AgNW with two sizes using two models, human primary keratinocytes and human reconstructed epidermis. In keratinocytes, AgNW are rapidly and massively internalized inside cells leading to dose-dependent cytotoxicity that was not due to Ag⁺ release. Analysing our data with different dose metrics, we propose that the number of NW is the most appropriate dose-metric for studies of AgNW toxicity. In reconstructed epidermis, the results of a standard in vitro skin irritation assay classified AgNW as non-irritant to skin and we found no evidence of penetration into the deeper layer of the epidermis. The findings show that healthy and intact epidermis provides an effective barrier for AgNW, although the study does not address potential transport through follicles or injured skin. The combined cell and tissue model approach used here is likely to provide an important methodology for assessing the risks for skin exposure to AgNW from consumer products.

UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins.

Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.

Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures.

Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd.

Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.

Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT-PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin-EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing.

Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.

JNK regulates compliance-induced adherens junctions formation in epithelial cells and tissues.

We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1(-/-) or jnk2(-/-) mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo.

Effects of THz Exposure on Human Primary Keratinocyte Differentiation and Viability.

Primary human keratinocytes can be driven,in vitro, to differentiate, viaactivation of transglutaminases, by raisingthe culture medium calcium concentrationabove 1 mM. This results intransglutaminase regulated cross linking ofspecific amino acids with resultantcornified envelope formation. Thedifferentiation was monitored via theincorporation of fluorescein cadaverineinto the cornified envelops. Thisdifferentiation assay was combined withassessment of reductive capacity ofresazurin, as a measure of cellactivity/viability.One primary aim is to assess the effects ofTHz radiation on human skin, since medicalimaging of the body through the skin isenvisaged.Human keratinocytes, at passage 2 fromisolation, were grown to confluence, andtransported in a buffered salt solution at22 (°)C. The exposure to the THz sourcewas for 10, 20 or 30 minutes at roomtemperature.No donor specific inhibition or stimulationof cell activity, compared with non-exposedcells, was noted following exposure in therange 1 to 3 THz, at up to 0.45J/cm(2).The differentiation also occurred in anormal way, for exposed and non-exposedcells, with the FC incorporation increasingbetween day 3 and day 8, as previouslynoted.