Skin substitutes based on gellan gum with mechanical and penetration compatibility to native human skin.

The study reports on a simple system to fabricate skin substitutes consisting of a naturally occurring bacterial polysaccharide gellan gum. Gelation was driven by the addition of a culture medium whose cations induced gellan gum crosslinking at physiological temperature, resulting in hydrogels. Human dermal fibroblasts were incorporated in these hydrogels and their mechanical, morphological, and penetration characteristics were studied. The mechanical properties were determined by means of oscillatory shear rheology, and a short linear viscoelastic regime was noted up to less than 1% of strain amplitude. The storage modulus increased with an increasing polymer concentration. The moduli were in the range noted for native human skin. After 2 weeks of fibroblast cultivation, the storage moduli showed signs of deterioration, so that a culture time of 2 weeks was proposed for further studies. Microscopic and fluorescent staining observations were documented. These depicted a crosslinked network structure in the hydrogels with a homogeneous distribution of cells and an assured cell viability of 2 weeks. H&E staining was also performed, which showed some traces of ECM formation in a few sections. Finally, caffeine penetration experiments were carried out with Franz diffusion cells. The hydrogels with a higher concentration of polymer containing cells showed an improved barrier function against caffeine compared to previously studied multicomponent hydrogels as well as commercially available 3D skin models. Therefore, these hydrogels displayed both mechanical and penetration compatibility with the ex vivo native human skin.

TNF ΔARE Pigs: A Translational Crohn's Disease Model.

BACKGROUND AND AIMS: Crohn's disease [CD] is a major subtype of inflammatory bowel diseases [IBD] with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the pig has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. METHODS: We generated pigs bearing a 93-bp deletion of the adenosine-uracil-rich element [ARE] and a constitutive-decay element within the 3' untranslated region of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNFΔARE/+ and TNFΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. RESULTS: TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration and dysbiotic microbial communities. 16S rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. Principal components analysis clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. CONCLUSIONS: We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.

A humanized minipig model for the toxicological testing of therapeutic recombinant antibodies.

The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal models of human diseases are often unsuitable for immunogenicity testing and for predicting adverse outcomes in human patients. Most human therapeutic antibodies trigger xenogeneic responses in wild-type animals and thus rapid clearance of the drugs, which makes in vivo toxicological testing of human antibodies challenging. Here we report the generation of Göttingen minipigs carrying a mini-repertoire of human genes for the immunoglobulin heavy chains γ1 and γ4 and the immunoglobulin light chain κ. In line with observations in human patients, the genetically modified minipigs tolerated the clinically non-immunogenic IgG1κ-isotype monoclonal antibodies daratumumab and bevacizumab, and elicited antibodies against the checkpoint inhibitor atezolizumab and the engineered interleukin cergutuzumab amunaleukin. The humanized minipigs can facilitate the safety and efficacy testing of therapeutic antibodies.

Tumor Targeting with Bacterial Shiga Toxin B Subunit in Genetic Porcine Models for Colorectal Cancer and Osteosarcoma.

The B subunit of bacterial Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the tumor-targeting potential of STxB. In accordance with findings on human colorectal cancer, the pig model APC1311 bound STxB in colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for osteosarcoma, a tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig osteosarcoma strongly bound StxB and expressed the Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human osteosarcoma cell lines expressed A4GALT mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based tumor targeting and may allow "reverse-translational" predictions on human tumor biology.

The Missing Link: Cre Pigs for Cancer Research.

The Cre/loxP system is a powerful tool for the generation of animal models with precise spatial and temporal gene expression. It has proven indispensable in the generation of cancer models with tissue specific expression of oncogenes or the inactivation of tumor suppressor genes. Consequently, Cre-transgenic mice have become an essential prerequisite in basic cancer research. While it is unlikely that pigs will ever replace mice in basic research they are already providing powerful complementary resources for translational studies. But, although conditionally targeted onco-pigs have been generated, no Cre-driver lines exist for any of the major human cancers. To model human pancreatic cancer in pigs, Cre-driver lines were generated by CRISPR/Cas9-mediated insertion of codon-improved Cre (iCre) into the porcine PTF1A gene, thus guaranteeing tissue and cell type specific function which was proven using dual fluorescent reporter pigs. The method used can easily be adapted for the generation of other porcine Cre-driver lines, providing a missing tool for modeling human cancers in large animals.

Role of Methylation in Period2 (PER2) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical-Studies on the Pig Model.

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene (PER2), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma.

Osteosarcoma (OS) is a primary bone malignancy that mainly occurs during adolescent growth, suggesting that bone growth plays an important role in the aetiology of the disease. Genetic factors, such as heritable mutations of Rb1 and TP53, are associated with an increased risk of OS. Identifying driver mutations for OS has been challenging due to the complexity of bone growth-related pathways and the extensive intra-tumoral heterogeneity of this cancer. We previously generated pigs carrying a mutated TP53 gene, which develop OS at high frequency. RNA sequencing and allele expression imbalance (AEI) analysis of OS and matched healthy control samples revealed a highly significant AEI (p = 2.14 × 10-39) for SNPs in the BIRC3-YAP1 locus on pig chromosome 9. Analysis of copy number variation showed that YAP1 amplification is associated with the AEI and the progression of OS. Accordingly, the inactivation of YAP1 inhibits proliferation, migration, and invasion, and leads to the silencing of TP63 and reconstruction of p16 expression in p53-deficient porcine OS cells. Increased p16 mRNA expression correlated with lower methylation of its promoter. Altogether, our study provides molecular evidence for the role of YAP1 amplification in the progression of p53-dependent OS.

A tissue- and gender-specific regulation of the SARS-CoV-2 receptor ACE2 by p53 in pigs.

The current COVID-19 pandemic is caused by infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A sex-bias has been observed, with increased susceptibility and mortality in male compared to female patients. The gene for the SARS-CoV-2 receptor ACE2 is located on the X chromosome. We previously generated TP53 mutant pigs that exhibit a sex-specific patho-phenotype due to altered regulation of numerous X chromosome genes. In this study, we explored the effect of p53 deficiency on ACE2 expression in pigs. First, we identified the p53 binding site in the ACE2 promoter and could show its regulatory effect on ACE2 expression by luciferase assay in porcine primary kidney fibroblast cells. Later, quantitative PCR and western blot showed tissue- and gender-specific expression changes of ACE2 and its truncated isoform in p53-deficient pigs. We believe these findings will broaden the knowledge on ACE2 regulation and COVID-19 susceptibility.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA.

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

Polymorphisms of CSF1R and WISP1 genes are associated with severity of familial adenomatous polyposis in APC1311 pigs.

Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.

The expression of TAP1 candidate gene, but not its polymorphism and methylation, is associated with colonic polyp formation in a porcine model of human familial adenomatous polyposis.

In humans, the dysfunction of the adenomatous polyposis coli (APC) gene causes hereditary familial adenomatous polyposis (FAP) and increased risk of colorectal cancer (CRC). The severity of polyposis varies between individuals, but genetic basis for this is in large part unknown. This variability also occurs in our porcine model of FAP, based on an APC1311 mutation (orthologous to human APC1309). Since loss of TAP1 function can lead to CRC in humans, we searched for germline polymorphisms in APC1311/+ pigs with low (LP) and high (HP) levels of polyposis, as well as in wild-type pigs representing six breeds and a commercial line. The distribution of 40 identified polymorphic variants was similar in the LP and HP pigs. In contrast, the TAP1 transcript level was significantly higher in normal colon mucosa of HP pigs than in LP pigs. Moreover, six SNPs showed significant effects on TAP1 promoter activity, but no correlation with severity of polyposis was observed. Analysis of DNA methylation in the promoter region showed that one CpG site differed significantly between LP and HP pigs. We conclude that TAP1 genotype may not itself be associated with polyposis, but our findings concerning its expression suggest a role in the development of polyps.

Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis.

Early and comprehensive endoscopic detection of colonic dysplasia - the most clinically significant precursor lesion to colorectal adenocarcinoma - provides an opportunity for timely, minimally-invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) is described that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis. FSNs are biodegradable (t1/2 of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm2 with high tumor-to-background ratios. Furthermore, in the human-scale, APC 1311/+ porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100-150-nm) or composition (i.e., silica, silica/gold hybrid) have already been successfully translated to the clinic, and, clinical fluorescent/white light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC1311/+; orthologous to human APC1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5'-flanking, coding, and 3' untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p = 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.

Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus.

BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

Altered microRNA profiles during early colon adenoma progression in a porcine model of familial adenomatous polyposis.

MicroRNAs are dysregulated in various cancers including colorectal cancer, and are potential useful biomarkers of disease development. We used next generation sequencing to investigate miRNA expression profiles in low- and high-grade intraepithelial dysplastic polyps from pigs carrying a mutation in the adenomatous polyposis coli tumour suppressor (APC1311 , orthologous to human APC1309 ) that model an inherited predisposition to colorectal cancer, familial adenomatous polyposis. We identified several miRNAs and their isomiRs significantly (P < 0.05) differentially expressed between low and high-grade intraepithelial dysplastic polyps. Of these, ssc-let-7e, ssc-miR-98, ssc-miR-146a-5p, ssc-miR-146b, ssc-miR-183 and ssc-miR-196a were expressed at higher level and ssc-miR-126-3p at lower level in high-grade intraepithelial dysplastic polyps. Functional miRNA target analysis revealed significant (P < 0.001) over-representation of cancer-related pathways, including 'microRNAs in cancer', 'proteoglycans in cancer', 'pathways in cancer' and 'colorectal cancer'. This is the first study to reveal miRNAs associated with premalignant transformation of colon polyps.

Porcine familial adenomatous polyposis model enables systematic analysis of early events in adenoma progression.

We compared gene expression in low and high-grade intraepithelial dysplastic polyps from pigs carrying an APC 1311 truncating mutation orthologous to human APC 1309 , analysing whole samples and microdissected dysplastic epithelium. Gene set enrichment analysis revealed differential expression of gene sets similar to human normal mucosa versus T1 stage polyps. Transcriptome analysis of whole samples revealed many differentially-expressed genes reflecting immune infiltration. Analysis of microdissected dysplastic epithelium was markedly different and showed increased expression in high-grade intraepithelial neoplasia of several genes known to be involved in human CRC; and revealed possible new roles for GBP6 and PLXND1. The pig model thus facilitates analysis of CRC pathogenesis.