Characterization of heterozygous and homozygous mouse models with the most common hypertrophic cardiomyopathy mutation MYBPC3c.2373InsG in the Netherlands.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the cardiac myosin binding protein-C (cMyBP-C) encoding gene MYBPC3. In the Netherlands, approximately 25% of patients carry the MYBPC3c.2373InsG founder mutation. Most patients are heterozygous (MYBPC3+/InsG) and have highly variable phenotypic expression, whereas homozygous (MYBPC3InsG/InsG) patients have severe HCM at a young age. To improve understanding of disease progression and genotype-phenotype relationship based on the hallmarks of human HCM, we characterized mice with CRISPR/Cas9-induced heterozygous and homozygous mutations. At 18-28 weeks of age, we assessed the cardiac phenotype of Mybpc3+/InsG and Mybpc3InsG/InsG mice with echocardiography, and performed histological analyses. Cytoskeletal proteins and cardiomyocyte contractility of 3-4 week old and 18-28 week old Mybpc3c.2373InsG mice were compared to wild-type (WT) mice. Expectedly, knock-in of Mybpc3c.2373InsG resulted in the absence of cMyBP-C and our 18-28 week old homozygous Mybpc3c.2373InsG model developed cardiac hypertrophy and severe left ventricular systolic and diastolic dysfunction, whereas HCM was not evident in Mybpc3+/InsG mice. Mybpc3InsG/InsG cardiomyocytes also presented with slowed contraction-relaxation kinetics, to a greater extent in 18-28 week old mice, partially due to increased levels of detyrosinated tubulin and desmin, and reduced cardiac troponin I (cTnI) phosphorylation. Impaired cardiomyocyte contraction-relaxation kinetics were successfully normalized in 18-28 week old Mybpc3InsG/InsG cardiomyocytes by combining detyrosination inhibitor parthenolide and ß-adrenergic receptor agonist isoproterenol. Both the 3-4 week old and 18-28 week old Mybpc3InsG/InsG models recapitulate HCM, with a severe phenotype present in the 18-28 week old model.

Drug Metabolism of Hepatocyte-like Organoids and Their Applicability in In Vitro Toxicity Testing.

Emerging advances in the field of in vitro toxicity testing attempt to meet the need for reliable human-based safety assessment in drug development. Intrahepatic cholangiocyte organoids (ICOs) are described as a donor-derived in vitro model for disease modelling and regenerative medicine. Here, we explored the potential of hepatocyte-like ICOs (HL-ICOs) in in vitro toxicity testing by exploring the expression and activity of genes involved in drug metabolism, a key determinant in drug-induced toxicity, and the exposure of HL-ICOs to well-known hepatotoxicants. The current state of drug metabolism in HL-ICOs showed levels comparable to those of PHHs and HepaRGs for CYP3A4; however, other enzymes, such as CYP2B6 and CYP2D6, were expressed at lower levels. Additionally, EC50 values were determined in HL-ICOs for acetaminophen (24.0−26.8 mM), diclofenac (475.5−>500 µM), perhexiline (9.7−>31.5 µM), troglitazone (23.1−90.8 µM), and valproic acid (>10 mM). Exposure to the hepatotoxicants showed EC50s in HL-ICOs comparable to those in PHHs and HepaRGs; however, for acetaminophen exposure, HL-ICOs were less sensitive. Further elucidation of enzyme and transporter activity in drug metabolism in HL-ICOs and exposure to a more extensive compound set are needed to accurately define the potential of HL-ICOs in in vitro toxicity testing.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7.

BACKGROUND: Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level. RESULTS: We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes. CONCLUSIONS: We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of the gene. The mutated gene implicates the extracellular matrix in the regular organization of chrondrocyte columns of the growth plate. Conservation of individual amino acids may not be necessary at the protein level but instead may reflect underlying conservation of nucleotide sequence that are required for efficient splicing.

A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses.

BACKGROUND: Hydrocephalus in Friesian horses is a developmental disorder that often results in stillbirth of affected foals and dystocia in dams. The occurrence is probably related to a founder effect and inbreeding in the population. The aim of our study was to find genomic associations, to investigate the mode of inheritance, to allow a DNA test for hydrocephalus in Friesian horses to be developed. In case of a monogenic inheritance we aimed to identify the causal mutation. RESULTS: A genome-wide association study of hydrocephalus in 13 cases and 69 controls using 29,720 SNPs indicated the involvement of a region on ECA1 (P <1.68 × 10(-6)). Next generation DNA sequence analysis of 4 cases and 6 controls of gene exons within the region revealed a mutation in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) as the likely cause of hydrocephalus in Friesian horses. The nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* was identical to a B3GALNT2 mutation identified in a human case of muscular dystrophy-dystroglycanopathy with hydrocephalus. All 16 available cases and none of the controls were homozygous for the mutation, and all 17 obligate carriers (= dams of cases) were heterozygous. A random sample of the Friesian horse population (n = 865) was tested for the mutation in a commercial laboratory. One-hundred and forty-seven horses were carrier and 718 horses were homozygous for the normal allele; the estimated allele frequency in the Friesian horse population is 0.085. CONCLUSIONS: Hydrocephalus in Friesian horses has an autosomal recessive mode of inheritance. A nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* in B3GALNT2 (1:75,859,296-75,909,376) is concordant with hydrocephalus in Friesian horses. Application of a DNA test in the breeding programme will reduce the losses caused by hydrocephalus in the Friesian horse population.

Pulmonary alveolar proteinosis in a cat.

BACKGROUND: Pulmonary alveolar proteinosis is an extremely rare lung disease in animals and humans. It is characterized by the deposition of a large amount of phospholipoproteinaceous material in the alveoli. There are several possible etiologies, both congenital and acquired. Alveolar macrophages play an important role in the clearance of surfactant. This is the first report of pulmonary alveolar proteinosis in the feline species. CASE PRESENTATION: Pulmonary alveolar proteinosis was diagnosed in an 8-month-old cat with chronic tachypnea, failure to thrive and finally respiratory distress. The diagnosis was based on the milky appearance of the bronchoalveolar lavage fluid taken under general anesthesia after bronchoscopy. Because of the worsening respiratory distress and development of anorexia the kitten was euthanized. Histopathology of the lungs showed alveoli and bronchi filled with eosinophilic material. Electron microscopy revealed lamellated intra-alveolar bodies. As the granulocyte-macrophage colony-stimulating factor was elevated in the serum and no autoantibodies against granulocyte-macrophage colony-stimulating factor were detected, a primary hereditary pulmonary alveolar proteinosis was suspected. The underlying cause was thought to be a dysfunction of the receptor of the granulocyte-macrophage colony-stimulating factor, however, a mutation in the genes encoding the alpha and beta chains of this receptor has not been found. CONCLUSION: This is the first description of pulmonary alveolar proteinosis in a cat. This kitten is thought to have a primary hereditary pulmonary alveolar proteinosis with a possible defect in the signalling pathway of the receptor of the granulocyte-macrophage colony-stimulating factor. The imaging and pathologic findings are similar to those of humans.

Genome-wide survey indicates involvement of loci on canine chromosomes 7 and 31 in patellar luxation in Flat-Coated Retrievers.

BACKGROUND: Patellar luxation is an orthopedic disorder in which the patella moves out of its normal location within the femoral trochlea of the knee and it can lead to osteoarthritis, lameness, and pain. In dogs it is a heritable trait, with both environmental and genetic factors contributing to the phenotype. The prevalence of patellar luxation in the Dutch Flat-Coated Retriever population is 24%. In this study, we investigated the molecular genetics of the disorder in this population. RESULTS: Genome-wide association analysis of 15,823 single nucleotide polymorphisms (SNPs) in 45 cases and 40 controls revealed that patellar luxation was significantly associated with a region on chromosome CFA07, and possibly with regions on CFA03, CFA31, and CFA36. The exons of the genes in these regions, 0.5 Mb combined, were analyzed further. These exons from 15 cases and a pooled sample from 15 controls were enriched using custom genomic hybridization arrays and analyzed by massive parallel DNA sequencing. In total 7257 variations were detected. Subsequently, a selection of 144 of these SNPs were genotyped in 95 Flat-Coated Retrievers. Nine SNPs, in eight genes on CFA07 and CFA31, were associated with patellar luxation (P <10-4). Genotyping of these SNPs in samples from a variety of breeds revealed that the disease-associated allele of one synonymous SNP in a pseudogene of FMO6 was unique to Flat-Coated Retrievers. CONCLUSION: Genome-wide association analysis followed by targeted DNA sequencing identified loci on chromosomes 7 and 31 as being involved in patellar luxation in the Flat-Coated Retriever breed.

Targeting lipid metabolism as a new therapeutic strategy for inherited cardiomyopathies.

Inherited cardiomyopathies caused by pathological genetic variants include multiple subtypes of heart disease. Advances in next-generation sequencing (NGS) techniques have allowed for the identification of numerous genetic variants as pathological variants. However, the disease penetrance varies among mutated genes. Some can be associated with more than one disease subtype, leading to a complex genotype-phenotype relationship in inherited cardiomyopathies. Previous studies have demonstrated disrupted metabolism in inherited cardiomyopathies and the importance of metabolic adaptations in disease onset and progression. In addition, genotype- and phenotype-specific metabolic alterations, especially in lipid metabolism, have been revealed. In this mini-review, we describe the metabolic changes that are associated with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which account for the largest proportion of inherited cardiomyopathies. We also summarize the affected expression of genes involved in fatty acid oxidation (FAO) in DCM and HCM, highlighting the potential of PPARA-targeting drugs as FAO modulators in treating patients with inherited cardiomyopathies.

A novel IBA57 variant is associated with mitochondrial iron-sulfur protein deficiency and necrotizing myelopathy in dogs.

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case-control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5 Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron-sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57-depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants.

Locational memory of macrovessel vascular cells is transcriptionally imprinted.

Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems.

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target.

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%-20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log2 fold change was >2 or < -2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans.

Whole transcriptome analysis of canine pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies. To identify druggable pathways driving PPGL development, we performed RNA sequencing on PPGLs (n = 19) and normal adrenal medullas (NAMs; n = 10) of dogs. Principal component analysis (PCA) revealed that PPGLs clearly clustered apart from NAMs. In total, 4,218 genes were differentially expressed between PPGLs and NAMs. Of these, 232 had a log2 fold change of >3 or < -3, of which 149 were upregulated in PPGLs, and 83 were downregulated. Compared with NAMs, PPGLs had increased expression of genes related to the cell cycle, tumor development, progression and metastasis, hypoxia and angiogenesis, and the Wnt signaling pathway, and decreased expression of genes related to adrenal steroidogenesis. Our data revealed several overexpressed genes that could provide targets for novel therapeutics, such as Ret Proto-Oncogene (RET), Dopamine Receptor D2 (DRD2), and Secreted Frizzled Related Protein 2 (SFRP2). Based on the PCA, PPGLs were classified into 2 groups, of which group 1 had significantly higher Ki67 scores (p = 0.035) and shorter survival times (p = 0.04) than group 2. Increased expression of 1 of the differentially expressed genes between group 1 and 2, pleiotrophin (PTN), appeared to correlate with a more aggressive tumor phenotype. This study has shed light on the transcriptomic profile of canine PPGL, yielding new insights into the pathogenesis of these tumors in dogs, and revealed potential novel targets for therapy. In addition, we identified 2 transcriptionally distinct groups of PPGLs that had significantly different survival times.

Identification of novel genetic risk factors of dilated cardiomyopathy: from canine to human.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. METHODS: We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. RESULTS: Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. CONCLUSIONS: Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein.

Congenital disorders of the hepatic portal vasculature are rare in man but occur frequently in certain dog breeds. In dogs, there are two main subtypes: intrahepatic portosystemic shunts, which are considered to stem from defective closure of the embryonic ductus venosus, and extrahepatic shunts, which connect the splanchnic vascular system with the vena cava or vena azygos. Both subtypes result in nearly complete bypass of the liver by the portal blood flow. In both subtypes the development of the smaller branches of the portal vein tree in the liver is impaired and terminal branches delivering portal blood to the liver lobules are often lacking. The clinical signs are due to poor liver growth, development, and function. Patency of the ductus venosus seems to be a digenic trait in Irish wolfhounds, whereas Cairn terriers with extrahepatic portosystemic shunts display a more complex inheritance. The genes involved in these disorders cannot be identified with the sporadic human cases, but in dogs, the genome-wide study of the extrahepatic form is at an advanced stage. The canine disease may lead to the identification of novel genes and pathways cooperating in growth and development of the hepatic portal vein tree. The same pathways likely regulate the development of the vascular system of regenerating livers during liver diseases such as hepatitis and cirrhosis. Therefore, the identification of these molecular pathways may provide a basis for future proregenerative intervention.

Distribution of extrahepatic congenital portosystemic shunt morphology in predisposed dog breeds.

BACKGROUND: An inherited basis for congenital extrahepatic portosystemic shunts (EHPSS) has been demonstrated in several small dog breeds. If in general both portocaval and porto-azygous shunts occur in breeds predisposed to portosystemic shunts then this could indicate a common genetic background. This study was performed to determine the distribution of extrahepatic portocaval and porto-azygous shunts in purebred dog populations. RESULTS: Data of 135 client owned dogs diagnosed with EHPSS at the Faculty of Veterinary Medicine of Utrecht University from 2001 - 2010 were retrospectively analyzed. The correlation between shunt localization, sex, age, dog size and breed were studied. The study group consisted of 54 males and 81 females from 24 breeds. Twenty-five percent of dogs had porto-azygous shunts and 75% had portocaval shunts. Of the dogs with porto-azygous shunts only 27% was male (P = 0.006). No significant sex difference was detected in dogs with a portocaval shunt. Both phenotypes were present in almost all breeds represented with more than six cases. Small dogs are mostly diagnosed with portocaval shunts (79%) whereas both types are detected. The age at diagnosis in dogs with porto-azygous shunts was significantly higher than that of dogs with portocaval shunts (P < 0.001). CONCLUSION: The remarkable similarity of phenotypic variation in many dog breeds may indicate common underlying genes responsible for EHPSS across breeds. The subtype of EHPSS could be determined by a minor genetic component or modulating factors during embryonic development.

Genetic Basis of Dilated Cardiomyopathy in Dogs and Its Potential as a Bidirectional Model.

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment.

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do.

Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels.

Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.