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Abstract

Swine are increasingly studied as animal models of human disease. The anatomy, size, longevity, physiology, immune system, and metabolism of swine are more like humans than traditional rodent models. In addition, the size of swine is preferred for surgical placement and testing of medical devices destined for humans. These features make swine useful for biomedical, pharmacological, and toxicological research. With recent advances in gene-editing technologies, genetic modifications can readily and efficiently be made in swine to study genetic disorders. In addition, gene-edited swine tissues are necessary for studies testing and validating xenotransplantation into humans to meet the critical shortfall of viable organs versus need. Underlying all of these biomedical applications, the knowledge of husbandry, background diseases and lesions, and biosecurity needs are important for productive, efficient, and reproducible research when using swine as a human disease model for basic research, preclinical testing, and translational studies.

Keywords

animal models genetic disease medical devices pig diseases swine xenotransplantation

Swine were domesticated over 10,000 years ago in the Near East and have continued to be a species closely linked with human existence.³² In fact, during the Shang dynasty (approximately 1600–1050 years before the common era), the symbol for home or house was depicted as a pig with a roof over it.¹⁴⁴

The first well-documented experiments involving pigs were performed more than 2000 years ago, by Erasistratus, Galen, and Aristotle.^54,147 From these investigations, a base understanding of the anatomic and physiologic relationships between the heart, blood flow, vasculature, and lungs was established. Galen also studied the nervous system in pigs and demonstrated the necessity of the recurrent laryngeal nerve for vocalization. He also showed that transection of the spinal cord between the third and fourth vertebra resulted in the cessation of respiration in the pig.^54,147

Swine have been studied for many years, but the popularity of swine in research has dramatically increased in recent years for several reasons. First, the pig is closer in size and has an increased lifespan more comparable to humans than most other research species, making it a useful model for many preclinical and translational studies. These factors are especially important for studies in trauma, medical imaging (diagnostic, therapeutic, monitoring), cancer, and surgical interventions, to name a few.^{71,103,119,130} Second, traditional rodent models do not always produce relevant phenotypes for studying some conditions or for preclinical testing and safety assessments. Swine are a suitable large animal model that can fill in this gap in model biology. Examples of this include cystic fibrosis (CF) and Duchenne muscular dystrophy.^57,72,115 Third, recent development of new gene editing techniques have accelerated the efficiency and speed of generating swine models using targeted gene-editing.^143,150 Fourth, limitations in swine-specific expertise, reagents, and facilities had been a concern for investigators, but these hurdles are being removed making swine models more attractive and feasible for translational studies.¹⁰ Finally, swine have preferential advantages over other traditional large animal models. The use of nonhuman primates (NHPs) is increasingly restricted by ethical issues, costs, and increased regulations.¹⁰ Use of canine species has also been on the decline in recent years. For example, dogs are considered a sensitive species and special approvals are required before their use as subjects in funded research by the United States Veterans Administration.⁸⁵ With the looming concerns of using NHP and dog models, swine have become an appropriate option for large animal studies. In addition, pigs are increasingly being used in pharmacological studies to examine pharmacokinetics, oral bioavailability, skin penetrance, and toxicology of new drugs and entities, the discussion of which is beyond the scope of this article.^7,46,110,132

Numerous swine breeds are available,¹³⁰ and similar to dog breeds, were often created to serve specific purposes. Agricultural breeds such as the Yorkshire, Duroc, and Landrace were bred for feed efficiency and high fecundity. Agricultural breeds have also been bred for meat quality, which has resulted in selecting for or against some cytochromes which may affect toxicology studies.¹⁹ There are over 30 breeding groups of minipigs in the world.¹¹⁶ Those discussed here are most commonly represented in the literature. The most popular minipig breeds used in research in the United States are the Göttingen, Hanford, Sinclair, and Yucatan. Both the Sinclair and Yucatan have mini or micro lines that have been selected for decreased size to facilitate toxicology studies with smaller amounts of test article requirements and to control the need for larger housing units. The Göttingen and the micro-Yucatan are the smallest of the minipig breeds. The Hanford is most similar in size to a human and is therefore well-suited to surgical studies that require any subsequent length of survival. The Göttingen is also available in Europe and Asia, but these are separate breeding colonies and genetic divergence has occurred. In Japan, the Micromini breed is popular, and in China, there is an inbred breed called Bama. Apart from the Bama breed, all other breeds mentioned here are considered outbred. The terminology of breed versus strain in pigs has not yet become stringently defined. In mice, a strain is defined as a line that is genetically identical resulting from repeated brother × sister matings (typically 20 generations). In pigs, this has only been pursued in a few breeds (e.g. Bama, Wuzhishan) with the Wuzhishan breed having both inbred and outbred lines that have been developed.^80,142,153 Additional resources about pig breeds and resources are available for further reading.^40,116,130

Other considerations include the source of the swine. Many Yorkshire background pigs in the United States originate from farms that also breed animals for market. Although housing facilities for agricultural and research animals may be separate, animals from these vendors may be exposed to the typical swine diseases.^45,155 Research facilities and equipment are also important considerations. For instance, swine surgeries or imaging may require dedicated facilities, equipment, and professional expertise for successful studies.^118,130

Common Nutritional and Infectious Diseases of Swine That May Impact Biomedical Studies

Health challenges in commercially raised pigs often present a diagnostic dilemma and potential complication in research studies.^45,155 Many common pathogens are endemic on commercial farms, so disease expression depends upon the interplay of host susceptibility (immunity, nutrition, and genetics), environmental factors (temperature, humidity, animal density, etc.), and the pathogen itself (dose and virulence). Given this interplay, disease expression is often variable within and among farms or vendors. From a diagnostic standpoint, detection of a pathogen may or may not equal disease, and it is up to the clinician, diagnostician, or both to align diagnostic test results with clinical and pathological observations.

Clinical signs, history, and epidemiologic observations are important components for generating a specific case definition in a population of pigs. The case definition is a critical step in the diagnostic process. All deviations from normal (physiological or behavioral) are important clues used to differentiate disease processes and should be characterized, quantified, and recorded as part of daily clinical observations. When coupled with gross lesions at necropsy, these data drive the case definition and inform sampling for laboratory testing to screen for specific etiological agents. In pigs, many pathogens commonly affect pigs at consistent periods in the production cycle (suckling phase, weaning phase, grower-finisher, and adult), and this can also help in formulating a case definition. Table 1 provides a list of some of the more common infectious agents detected in U.S. commercial swine, along with most impacted ages, typical clinical signs, and microscopic lesions. It is important to remember that microscopic lesions might be observed when reviewing samples from a diagnostic case or when scoring samples for a research investigation.⁴⁵

Table 1.

Common endemic infectious diseases in U.S. swine.

Etiologic Agent	Age Often Affected	Clinical Signs	Microscopic Lesions
Enteric pathogens
Coronaviruses	All ages	Acute watery diarrhea, can be severe in neonates	Segmental villus blunting and fusion that can be severe; epithelial necrosis; mild inflammation
Clostridium perfringens type C	Birth to 7 days	Hemorrhagic diarrhea; rapid death often of an entire litter	Mucosal necrosis, suppuration, hemorrhage, emphysema; gram-positive rods; diphtheritic membrane
Clostridioides difficile	Birth to 2 weeks	Creamy diarrhea; dehydration; usually mild but can be severe if poor immunity	Erosive typhlocolitis with multifocal “volcano” lesions, suppuration, mesocolonic edema
Rotavirus	Birth to 6 weeks	Creamy diarrhea; dehydration; usually mild; decreased growth rate	Segmental villus blunting; epithelial necrosis; mild inflammation
E. coli	Birth to 7 weeks	Acute diarrhea, watery, severe; dehydration; neonates (1-7 days) or postweaning (3-7 weeks)	None; short bacterial rods attached to enterocytes; congestion
Coccidia	1-4 weeks	Creamy diarrhea; dehydration; usually mild; decreased growth rate	Villous atrophy, fibrinonecrotic enteritis, intracellular merozoites
Salmonella spp.	After 3 weeks	Diarrhea, mild to severe, may see fibrin flecks; fever	Ulceration and suppuration; colon often more severely affected
Lawsonia spp.	After 5 weeks	Diarrhea, intermittent or severe with blood, pallor, rapid death; decreased growth	Crypt enterocyte hyperplasia, branching crypts, loss of goblet cells; necrosis; argyrophilic curved rods within epithelial cells
Brachyspira spp.	After 5 weeks	Mucoid diarrhea, +/- blood (dysentery); dehydration; reduced gain; deaths	Erosion of epithelium; goblet-cell hyperplasia, mixed inflammatory infiltrates, fibrinous exudate
Respiratory Pathogens
Influenza A virus	After 2 weeks	Abrupt onset of fever, sneeze, cough; high morbidity; lethargy; oculonasal discharge	Necrotizing bronchitis and bronchiolitis; necrotic, suppurative plugs in terminal airways; peribronchiolar lymphocytic cuffing with chronicity
Mycoplasma hyopneumoniae	After 3 weeks	Cough, especially on arousal; escalates over time	Suppurative and histiocytic bronchopneumonia with bronchus-associated lymphoid tissue hyperplasia
Actinobacillus spp.	After 2 weeks	Dyspnea, acute, rapid prostration and death; epistaxis	Necrosis, hemorrhage, suppuration, streaming leukocytes and pleuritis
Secondary bacterial pneumonia (Bordetella, Glaesserella, Pasteurella, Streptococcus, Trueperella)	After 2 weeks	Cough, fever, expiratory dyspnea, lethargy; pneumonia of variable duration	Suppurative bronchopneumonia; abscesses and pleuritis possible
Systemic Pathogens
Porcine reproductive and respiratory syndrome virus	All ages	Dyspnea, fever, anorexia, poor growth	Lymphohistiocytic interstitial pneumonia; necrotic macrophages; occasionally nephritis, myocarditis, encephalitis
Systemic bacterial infection (e.g. Streptococcus suis, Glaesserella sp., Mycoplasma hyorhinis)	After 2 weeks	Acute, subacute, or long-term multisystemic disease; paddling leg movements; found dead	Fibrinopurulent serositis, septic thrombi, lesions in other tissue localizations
Porcine circovirus type 2	After 4 weeks	Acute, subacute, or long-term dyspnea; diarrhea; wasting; uremia, icterus, rapid deaths	Lymphoid depletion, granulomatous inflammation (lymph node, lung, intestine, kidney, liver)
Porcine circovirus type 3	After 6 weeks	Ill-thrift, nonspecific, may be subclinical	Lymphoplasmacytic periarteritis in heart, kidney, and mesentery

To further confound the issue, many available diagnostic tests (polymerase chain reaction [PCR], culture, enzyme-linked immunosorbent assay [ELISA], and some immunohistochemistry (IHC)) do not differentiate pathogenic strains of some endemic agents from nonpathogenic strains or attenuated vaccine strains. Modified live vaccines are common in swine production (porcine reproductive and respiratory syndrome virus [PRRSV], Lawsonia spp., Salmonella spp., and Escherichia coli), and their use and the time since administration must be accounted for when interpreting diagnostic test results. Finally, mixed infections with one or more endemic agents are common, if not the normal. Additional ancillary diagnostic techniques such as in situ hybridization and gene sequencing may be required to differentiate pathogens from nonpathogens.

When using commercial pigs in research, it is also important to have a relevant understanding of modern swine production. Domestic pigs raised in confinement are restricted to what is provided and many diets are prepared in small mills or mixed on site. In this way, there is opportunity for human error in diet preparation, and diet formulations are therefore not always equal to the “diet as fed.” Furthermore, commercial swine diets are generally formulated to minimize cost and use cereal grains as base ingredients with supplemental protein, vitamins, and minerals. As such, deficiencies and over-supplementation are possible. Much of the nutritional research for pigs is done in optimal environments, whereas pigs in endemically infected herds or under experimental conditions may have different metabolic demands and needs. Formulations are also based on the mean response in a population, and individual animals may show signs of deficiency while most of a population remains unaffected. It is good practice to save liver, serum, and feed samples in any research study involving pigs for potential future analysis should study results indicate the potential for underlying nutritional issues.

Common nutritional issues in commercial swine include deficiencies in vitamins A, D, and E and deficiencies in iron and selenium. Marginal deficiency in vitamin A is common and may manifest as poor reproductive performance, weak born piglets, and increased susceptibility to infections. Sow’s milk is iron deficient, so piglets require supplementation within 5 days of birth. Piglets not receiving proper supplementation may develop microcytic, hypochromic anemia. Mulberry heart disease (MHD) is a nutritional cardiomyopathy that is often observed in well-conditioned, young growing pigs and is thought to occur due to excess free radicals. While MHD has been historically associated with vitamin E and selenium deficiency, this condition can manifest in pigs sufficient in both vitamin E and selenium.⁸⁶ In one study involving samples received at a veterinary diagnostic laboratory, liver selenium concentrations were adequate in all pigs with lesions of MHD and vitamin E concentrations were only deficient in 25% of pigs with MHD.⁹² This discord has prompted investigation into other potential causes of MHD, such as viral coinfections, but evidence for coinfections has thus far been lacking.¹¹⁷ It is important to note that nutrient requirements in pigs can be interdependent and, as an example, several dietary factors (iron, copper sulfur, etc.) can influence vitamin E requirements.^47,48,120 Disorders in calcium and phosphorus homeostasis can occur secondary to diet formulation errors, including vitamin D deficiency, and can manifest in varying degrees of lameness and unexpected fractures.

While responsible animal research functions under the care of a clinical veterinarian, it is useful to include an experienced swine pathologist and diagnostic laboratory resources as part of the team. Awareness of background diseases optimizes biosecurity and management practices to prevent these conditions from confounding the reproducibility of research. Examples of potential background lesions that can confound research studies are shown in Fig. 1a–c.

Fig. 1.

Examples of incidental and model-specific lesions in swine. (a and b) A 2-month-old pig with histiocytic effacement of the bronchus-associated lymphoid tissue in (a) lung and (b) gut-associated lymphoid tissue (GALT) in ileum caused by porcine circovirus-2 infection. Note the botryoid cytoplasmic inclusions in macrophages of the GALT (b, inset). Hematoxylin and eosin (HE). (c) Stomach from a 2-month-old pig with ulceration of the pars esophagea and hemorrhage evidenced as black, coffee ground-like material on adjacent stomach mucosa. (d and e) Two-month-old pig model of cystic fibrosis (CF). (d) The airway is obstructed with mucopurulent secretions and (e) pancreas is composed of remnant lobules sitting in a backdrop of white adipose tissue. The cystic (*) and fibrotic regions of the diseased CF pancreas (e) were the original basis for naming the condition cystic fibrosis.^5,127 HE and Masson’s trichrome, respectively. (f) Newborn swine model of ectodermal dysplasia.⁹¹ Note that the tracheal wall has normal structure except for the absence of submucosal glands.

Medical Device Testing

Medical devices are an omnipresent feature of modern medical care. The potential locations and functions of device implants combined with their future applications appear limitless, and their mode of therapy delivery is broad and will most certainly expand to human diseases in ways we have yet to fathom. In contrast to the pharmaceutical industry in which progress is often painstakingly incremental, medical device innovation and improvement is very fast-paced and preclinical testing must be concluded before first use in humans. Preclinical studies are performed primarily in large animal species, such as pigs, sheep, goats, and decreasingly in dogs.⁸⁵ These studies are sandwiched between bench tests to ensure engineering performance and device longevity on one hand, and clinical trials to confirm safety and ensure efficacy on the other. Preclinical testing is a scientific necessity and a regulatory requirement to reveal if a device performs to its intended use and has the anticipated biological and physiological effects. The pathology portion of well-executed and well-documented preclinical research plays an important role in accelerating the approval process as it provides sound scientific evidence that strengthens other study endpoints to substantiate claims.

When preclinical testing is needed to achieve regulatory approval for an implanted or temporarily inserted medical device,^6,31 several factors need to be considered in view of the proposed animal model. These factors include similarities to human anatomy and physiology, comparable organ sizes allowing treatment with or implantation of actual size medical devices as they are intended for human clinical implant (i.e., the “final finished product” with the intended clinical design), reproducibility based on low genetic variation among individuals, and an understanding of unavoidable background lesions including the impact of long-term healing effects.

There are no strict regulatory guidelines on the species or the number of animals to be used to reach approval for a medical device, let alone the details of how the pathology shall be conducted.³¹ If researchers have established that an animal study is necessary, regulatory bodies expect that a specific and detailed scientific justification will be provided for the animal species and number of animals.³¹ One approach is to model the study according to previous work that used a certain animal species in support of a highly similar, successfully approved device. Another approach is to adopt the methods of a preclinical study that was previously conducted in that species and published in peer-reviewed literature.³¹ Overall, the animal species and its physiological attributes should provide “a test system that offers a best attempt at simulating the clinical setting.”^31,34 A comprehensive understanding of the benefits and limitations of different species and the setting in which the study will be conducted is of unquestionable importance. Despite these aspirations, it is noteworthy that preclinical safety or efficacy recommendations for devices are usually based on low animal numbers, on abbreviated implant-duration times, and on implant data from healthy animals. Animal testing is both expensive and time consuming, and while necessary, does not entirely replicate the complexities that influence the phenotypic and molecular diversity observed in human diseases. However, clinical trials in humans also have inherent limitations and are even more costly and time-consuming than animal studies. Thus, animal models that have similar anatomy and biology, like swine, are useful for certain preclinical studies in testing medical devices.¹¹² Therefore, project leaders should prudently complete their risk assessment before conducting and explaining animal studies.

In addition to the physiologic and pathologic challenges in extrapolating animal data to humans,¹⁵⁴ the medical device world is prone to carelessly use the term “animal models” thus fostering the belief that there is a repository of animal disease models that can be used during device development.¹¹² In contrast, there is a low incidence of naturally occurring pathologies described in large animals that can be applied as actual disease models during device development. In the pig, for example, human intervention by way of selective breeding has eliminated genes that increase disease susceptibility. Moreover, most domestic farm pigs are slaughtered at a young age (less than 6-months-old), precluding the detection of late onset diseases such as cancer.⁴⁰ Genetically engineered pigs are vital for gaining a proper understanding of disease mechanisms⁹⁵ but have yet to find their way into medical device innovation, testing, and approval.¹¹² Put simply, current animal models used in device research or regulatory submissions are imperfect anatomical and physiological proxies to the human species. They are predominantly implant or surgery stand-ins, with the main intent of the normal, healthy animal recipient being of adequate size to accommodate human-tailored devices for an extended period of time to show evidence of safety.

Swine are the major large animal species used in the preclinical testing of medical devices. The U.S. Food and Drug Administration (FDA) recognizes swine as a suitable model for biocompatibility testing and the safety assessment of implanted devices in a variety of organs and systems.^56,130 Coronary stents,^84,93,94 abdominal aortic aneurysm grafts,¹⁵⁴ prosthetic heart valves,¹³³ abdominal meshes,⁵³ and neurovascular devices¹²¹ are medical devices that are commonly tested in swine. In addition, temporarily inserted energy-based devices for renal denervation¹⁰⁸ or for cardiac,^124,126 lung,¹¹⁴ or liver ablations⁵⁰ are usually evaluated in swine.

Juvenile farm swine are the default model for nonsurvival surgical training classes.¹³⁰ The haptics of performing surgery cannot be replaced by mannequins or simulators, so a pig or pig tissue is commonly used in training for interventional catheter techniques and complex trauma and endoscopic procedures. Apart from implanter training, prototype devices are also widely tested in farm pigs via acute nonsurvival procedures.

For survival studies that extend beyond a few weeks, the rapid growth rate of commercial farm pigs usually precludes its participation; this is not only because handling them at body weights beyond 100 kg is a challenge but also that their rapid growth impacts the sizing ratio between the implant location and the device, particularly for implantable cardiac or vascular devices. Fully grown miniature swine offer the same physiological advantages as the farm pig model, but at the equivalent maturity they are much smaller and therefore, the preferred selection for long-term studies. Currently, a widely used miniature swine breed is the Yucatan minipig. Although available from various vendors, their phenotype and disposition remain relatively consistent.⁸⁷ One disadvantage in the use of Yucatan pigs is their high cost. Since they are specifically bred for research in relatively small numbers, the cost per animal is typically 3 times higher than for a regular farm swine, and 1.5 to 2 times higher than for a purpose-bred hound.

While swine are a preferred species for testing medical devices, it is important to mention other large animal species that might have advantages over swine. Sheep are preferentially used for the long-term implantation of subcutaneous electronic devices (cardiac- or neuro-stimulators, see example in Supplemental Figure S1) with or without absorbable antibacterial envelopes⁶⁶ as well as for the testing of complex lead systems designed for deep-brain stimulation,²⁶ or brain catheters that are used in focal brain ablation.⁶¹ For central-nervous studies, sheep are preferred to swine due to their favorable skull anatomy. Sheep have also been the historical model for prosthetic mitral valve research,¹¹¹ as adolescent sheep show a detrimental tendency for leaflet calcification similar to that observed in children, and sheep continue to be used for catheter-delivered, suture-less mitral valves.¹³⁸ Skin implants in ovine candidates are favored to the porcine or canine model as sheep are less prone to pocket infections and can accommodate more concurrently implanted devices over their larger body surface; the sheep heart is also used for leadless cardiac pacing devices.¹³⁹ Dogs were the historical model for developing and refining cardiac leads for pacing, defibrillation, or both including recent magnetic resonance imaging (MRI)-safety experiments but are currently in the process of being replaced by pigs or sheep. This is usually the case unless the project has already matured toward a pivotal GLP study where dogs continue to be the preferred species for purposes of regulatory submission.

Several recent publications offer helpful approaches on how to perform the preclinical pathology analysis of medical devices^{33,106,122,141} or discuss their assessment via ISO standard 10993-6.⁸⁸ Without exaggeration, pathology data provide the most relevant measure of local device healing and its systemic consequences. This information is essential in determining the overall impact, both positive and negative, of a medical device on the body. As previously stated by Rouselle and Wicks, “Preparing and processing medical device implants for evaluation is a relatively high-risk and high-dollar process in which studies get made and endpoints can be lost with no second chance.”¹⁰⁵

Swine are currently center stage in the process of bringing incrementally improved or completely novel medical devices to market. At what time and to what degree computer modeling or human “digital twins” will substitute for a living animal in preclinical research, or whether genetic swine models of human disease will have matured into a practical, economical, and validated translational platform for testing device efficacy and safety cannot be predicted.

Swine Models for the Study of Genetic Diseases

Genetic disorders have been classified into 3 basic categories. The first category is monogenic disorders in which mutations of a specific gene causes the condition. An example of a monogenic disorder is CF with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR).¹²⁸ The second category of genetic disease is chromosomal disorders where chromosomes are absent or duplicated. An example of a chromosomal disorder is trisomy 21 (Down syndrome), a condition with 3 copies of chromosome 21 instead of standard 2 copies. The third category is complex disorders in which multiple genes and/or environmental conditions contribute in a complex fashion to develop disease. For example, predisposition to cancer can involve germline and somatic gene mutations in combination with environmental factors like diet, smoking exposure, and so on.⁸ As our understanding of gene regulation and expression continues to grow, the complexity of genetic diseases may not so easily fit into a discrete classification system.^51,113 Monogenic disorders will be highlighted as a basic example of the usefulness of gene-targeted swine models.

A historic advantage of mouse models over other species in research had been the ability to target genes using homologous recombination and embryonic stem cells.⁴¹ This approach was not readily adaptable to other species, giving mice a favored status by investigators. However, new opportunities began to appear in the 1990s when Dolly the cloned sheep was generated using somatic cell nuclear transfer.¹⁴⁶ Since then, a variety of new approaches including gene-editing techniques have provided the ability to specifically target a gene and make these edits in a much faster and efficient manner.^30,143,150 These newer technical advances have contributed to the growing number of useful swine models used to study human diseases.⁶⁸

Swine models of monogenetic diseases are often generated by targeting a gene of interest (mutation or excision) to prevent production of a functional protein (a “knockout”). However, in some situations, study of a genetic disorders may require further nuances to this approach to optimally study the model. For instance, mutations in CFTR^-/- produced a knockout model of CF, one of the first reported swine models of a human genetic disease.¹⁰⁴ However, the CFTR^-/- swine model exhibited a severe intestinal obstruction (meconium ileus) phenotype that required surgical correction to permit postnatal study.¹²⁷ One explanation for the enhanced severity of the swine model was that CFTR mutations in humans often produced a protein that had partial function (hypomorph), whereas the swine model was a null without functional protein.^104,127,140 Efforts to target the gene corresponding to the most common mutation in humans (F508) produced a swine model with low level of protein function (~6% of wildtype).⁸⁹ This low level of functional protein was sufficient to predispose the lung to CF lung diseases, but insufficient to correct the severe intestinal obstruction that required surgical intervention.⁸⁹ To overcome the intestinal obstruction, another knockout model was produced that also had a CFTR transgene under the control of the fatty acid binding protein promotor for targeted intestinal expression of CFTR protein. This model mitigated the severe intestinal phenotype to allow for postnatal lung disease studies without the requirement of surgical intervention.¹²⁹

The CF swine model has arguably had some of the broadest impact in studying a monogenic disorder while targeting a wide variety of systems, organs, and tissues (Fig. 1d, e and Table 2).¹²⁸ The breadth of monogenic disorders studied in gene-edited swine continues to grow, and these specific models often identify niches for targeted studies to accelerate research and complement other models. For instance, a recently developed model of ectodermal dysplasia lacks submucosal glands in cartilaginous airways (Fig. 1f).⁹¹ This model will be useful for studying the relative contributions of submucosal gland secretions to host defense in normal lung as well as CF models.

Table 2.

Selected examples of monogenic disorders studied in gene-edited swine.

Disorder (gene)	Tissues/Targets for Study
Cystic fibrosis (CFTR)	Lung (inflammation/infection,^9,13,89,127 mucus,^73,90,148 mucociliary transport,^49,90 antimicrobial proteins,⁹⁶ airway development,^2,3,28,77,78 smooth muscle,^20,21 epithelial lineages,¹³⁴ airway surface liquid pH,^64,65,115 gene therapy^22,23), sinonasal cavity,^17,129 exocrine pancreas,^1,79,104,135 endocrine,^63,102,136 central/ peripheral nervous systems (CNS/PNS),¹⁰¹ hepatobiliary,^{79,135,151,152} gastrointestinal,^79,104,129 cardiovascular,³⁹ skeletal,¹⁴ and reproductive⁹⁸
Ataxia telangiectasia (ATM)	CNS/cognitive,¹¹ growth abnormalities,¹¹ ataxia,¹¹ thymus,¹² reproductive,¹² and immune system¹²
Duchenne muscular dystrophy (DMD)	Skeletal muscle,^57,83 cardiovascular,¹³¹ gastrointestinal¹⁵⁶
Li Fraumeni (TP53)	Tumorigenesis/cancer, imaging/pathology correlates¹¹⁸
Ectodermal dysplasia (EDA1)	Absence of airway submucosal glands (predisposition to lung infection), alopecia⁹¹
Neurofibromatosis-1 (NF1)	Tumorigenesis/cancer,¹⁴⁵ CNS/cognitive,¹⁴⁵ dermal (axillary freckling/café-au-lait macules),¹⁴⁵ allodynia/nociception,^55,145 imaging/pathology correlates,^137,145 sleep/rest/behavior,⁵⁵ growth abnormalities¹³⁷
Hemophilia B (F9)	Coagulopathy, gene therapy¹⁸

The use of swine models for the study of genetic diseases has facilitated the development/validation of several diagnostic tools and research approaches that have helped advance the available resources for swine models in research. These resources include immunolabeling validation,^29,74,75 prosection techniques,⁷⁶ allodynia/nociception assessments,⁵⁵ cognitive/memory assessments,¹⁴⁵ recording sleep/rest/behavior patterns,⁵⁵ and imaging modalities/techniques,^118,119,125 to name a few.

Swine in Xenotransplantation

Organ transplantation in humans is the best therapy available for virtually any form of end-stage organ failure. It is a life-saving treatment for patients at imminent risk of death from heart, lung, or liver failure. Kidney transplantation provides a life-lengthening survival benefit for those with end-stage kidney disease on dialysis. Transplantation can even be considered “life-giving” in the case of uterine transplant. Despite the clear benefits, transplantation has always been limited by the availability of donor organs.

This has been especially true in the case of kidney transplantation. The increasing prevalence of end-stage kidney disease has created a situation where, until very recently, the number of patients added to transplant waiting lists has exceeded the number of transplants performed in a given year.⁶² Thus, the wait has grown longer and longer. Sadly, many patients will either die or become too sick for transplantation before a donor kidney becomes available. Decades of efforts to increase both living and deceased organ donation have had a modest effect and have not been able to overcome this deficit. These ongoing issues have prompted a search for alternative sources for donor organs.

Xenotransplantation is the transplantation of an organ from one species to another. This contrasts with allotransplantation, in which the organ is transplanted between 2 individuals of the same species. The concept of using an animal as the donor organ source is not new. Very early experiments in transplantation often used animals as the donor source;¹⁶ however, the lack of understanding of immunology doomed all of these experiments. Once clinical transplantation was shown to be feasible in 1954 in twins,⁴³ there was renewed interest in xenotransplantation. At this point, NHPs were thought to be the preferred source, owing to their close genetic relationship to humans.^100,123 These clinical experiments also failed. It would take several decades of developing an understanding of immunology before significant improvement in outcomes was seen in preclinical models. During that time, it also became apparent that NHPs would not be a suitable source animal. The need for organs would quickly outstrip the ability of NHP populations to provide them, and there was increasing concerns about the ethical use of NHPs in medicine and research, which persists today.¹⁰

Pigs offered a suitable alternative to NHPs as the donor source. As pigs have been domesticated and bred for human consumption for millennia, we have a good understanding of the required husbandry, and there are fewer ethical concerns regarding their use. Pigs reach reproductive age relatively early, reproduce quickly, and produce large litters, allowing for the development of a large donor population. Pigs grow quickly and reach an appropriate size for donation to a human early in life, but also have relatively long lifespans, so there is the potential a donated organ could last a long time posttransplantation. Finally, pig renal function is like that of humans in most ways, with similar electrolyte concentrations, osmolality, and glomerular filtration rates.^42,107

Despite these advantages of pigs as organ donors, crossing the species barrier introduces some new immunologic challenges. Pig cells express surface glycoproteins that are absent in humans and other primates.⁶⁰ These glycoproteins act as carbohydrate antigens, akin to ABO blood groups. The major antigen, the alpha-Gal antigen,⁶⁹ is expressed by human gut flora.³⁶ This exposure leads to innate antibody formation and immunologic memory against the antigen.³⁵ Therefore, pig-to-human xenotransplantation is a case of immunologically incompatible transplantation, and grafts suffer from hyperacute rejection within minutes of reperfusion. These challenges can at least be partially overcome by genetic manipulation of the donor pigs to delete alpha-1,3-galactosyltransferase,⁵⁸ which generates alpha-Gal, and this has been made considerably easier with the development of CRISPR-Cas9 techniques.¹⁰⁹

Work in pig-to-NHP models of xenotransplantation has shown that making these genetic modifications can improve the outcomes of xenotransplantation and prevent hyperacute rejection.^25,59 Subsequent efforts over many years have led to the development of multigene-edited pigs in which all 3 major carbohydrate antigens have been knocked-out; 6 human regulatory genes have been inserted, and the growth hormone receptor gene has been knocked out (Table 3). However, as the genetic modifications of the donor pig have been fine-tuned toward the human immune system in anticipation of clinical translation, the donor pigs have become less-ideal donors for an NHP immune system, limiting progress in studying these preclinical models.^4,70,149

Table 3.

Genetic modifications used in pig-to-human or pig-to-nonhuman primate xenotransplantation.

Gene	Target Product	Purpose of Modification
Carbohydrate antigens (knockouts)
GGTA1	Alpha-1,3-galactosyltransferase	Prevent synthesis of alpha-Gal antigen⁹⁷
CMAH	Cytidine monophospho-N-acetylneuraminic acid hydroxylase	Prevent synthesis of N-glycolylneuraminic acid antigen⁸¹
B4GALNT2	Beta-1,4 N-acetylgalactosaminyltransferase 2	Prevent synthesis of B4Gal SD blood group antigen¹⁵
Complement regulators
CD46	Membrane cofactor protein (MCP/CD46)	Inhibits complement by binding C3b/C4b⁶⁷
CD55	Complement decay-accelerating factor (DAF/CD55)	Indirectly blocks formation of membrane attack complex²⁷
CD59	MAC-inhibitory protein (MAC-IP/protectin/CD59)	Blocks the membrane attack complex by inhibiting C9 polymerization⁴⁴
Anti-inflammatory genes
HMOX1	Heme oxygenase 1 (HO-1)	Inhibits production of inflammatory mediators²⁵
CD47	Integrin associated protein (IAP/CD47)	Suppresses macrophage response via binding with SIRP-alpha²⁵
Coagulation regulators
THBD	Thrombomodulin (TM)	Reduces platelet aggregation⁵²
PROCR	Endothelial protein C receptor (EPCR/CD201)	Enhances activation of protein C⁵²
Other
GHR	Growth hormone receptor	Knockout in pig donor reduces organ growth post-transplantation³⁷

This has led to the development of a human preclinical research model, known as the Parsons model. In this approach, a recently deceased human, declared dead by brain-death criteria but deemed unsuitable for organ donation, is used as the recipient of pig organs. This allows testing of the donor organs in a human recipient, with a human immune system, without risk to a living human. Two groups have reported experiments using this approach and have demonstrated that no hyperacute rejection occurs, as predicted by prospective cross-matching; the kidney xenografts produce urine; and no zoonotic infection or chimerism was detected.^82,99 The Parsons model is limited by the pathophysiology of brain death, and therefore no long-term outcomes will be achieved in this model. However, this remains a critical step toward clinical translation of xenotransplantation.

In January 2022, the FDA granted a compassionate use exception for use of a porcine heart for xenotransplantation into a living human. The heart functioned well until the patient expired for unclear reasons 60 days following transplantation.³⁸ This case is notable for both the success of the transplant, but also because the recipient was found to have detectable porcine cytomegalovirus about 3 weeks posttransplant. This highlights ongoing knowledge gaps that will need to be addressed for xenotransplantation to fully reach the clinic, including the biosafety of porcine-derived grafts, the function of a porcine kidney in a living human, long-term durability of the xenotransplant, and optimal immunosuppression strategies. These issues are also observed in human-to-human allotransplantation and are not inherently unique to cross-species transplantation. However, in the context of allotransplantation, we benefit from roughly 70 years of clinical experience. In contrast, xenotransplantation remains in its infancy, and ultimately these questions may only be answerable in a clinical trial in living humans.

In conclusion, swine have increasingly become a useful and complementary resource for biomedical research, device testing, and medical therapies through xenotransplantation. The use of good swine husbandry and biosecurity practices along with awareness of common background diseases helps to prevent confounding factors that cloud reproducible research.

Supplemental Material

sj-pdf-1-vet-10.1177_03009858231222235 – Supplemental material for Swine models in translational research and medicine

Supplemental material, sj-pdf-1-vet-10.1177_03009858231222235 for Swine models in translational research and medicine by David K. Meyerholz, Eric R. Burrough, Nicole Kirchhof, Douglas J. Anderson and Kristi L. Helke in Veterinary Pathology

Footnotes

Supplemental material for this article is available online.

Authors’ Note

Part of this material was presented as part of a half-day session at the 2022 ACVP meeting in Boston, MA, USA.

Author Contributions

All authors contributed to the planning, writing, and editing of this manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would like to acknowledge support from the NIH (grant nos. HL163556, HL152960, and HL09184), Cystic Fibrosis Foundation, United Therapeutics Corporation, Lung Biotechnology PBC, and Revivicor.

ORCID iDs

David K. Meyerholz

Eric R. Burrough

Nicole Kirchhof

References

Abu-El-Haija

Ramachandran

Meyerholz

, et al. Pancreatic damage in fetal and newborn cystic fibrosis pigs involves the activation of inflammatory and remodeling pathways. Am J Pathol. 2012;181:499–507.

Adam

Abou Alaiwa

Bouzek

, et al. Postnatal airway growth in cystic fibrosis piglets. J Appl Physiol (1985). 2017;123:526–533.

Adam

Michalski

Bauer

, et al. Air trapping and airflow obstruction in newborn cystic fibrosis piglets. Am J Respir Crit Care Med. 2013;188:1434–1441.

Adams

Kim

Martens

, et al. Xenoantigen deletion and chemical immunosuppression can prolong renal xenograft survival. Ann Surg. 2018;268:564–573.

Andersen

DH.

Pathology of cystic fibrosis. Ann NY Acad Sci. 1962;93:500–517.

European Animal Research Association. EU regulations on animal research. Date unknown. Accessed December 20, 2023. https://www.eara.eu/animal-research-law#:~:text=Scientists%20must%20use%20the%20species,specifically%20bred%20for%20research%20purposes.

Ayuso

Buyssens

Stroe

, et al. The neonatal and juvenile pig in pediatric drug discovery and development. Pharmaceutics. 2020;13:44.

Balmain

Gray

Ponder

The genetics and genomics of cancer. Nat Genet. 2003;33:238–244.

Bartlett

Ramachandran

Wohlford-Lenane

, et al. Newborn cystic fibrosis pigs have a blunted early response to an inflammatory stimulus. Am J Respir Crit Care Med. 2016;194:845–854.

10.

Beck

Meyerholz

DK.

Evolving challenges to model human diseases for translational research. Cell Tissue Res. 2020;380:305–311.

11.

Beraldi

Chan

Rogers

, et al. A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease. Hum Mol Genet. 2015;24:6473–6484.

12.

Beraldi

Meyerholz

Savinov

, et al. Genetic ataxia telangiectasia porcine model phenocopies the multisystemic features of the human disease. Biochim Biophys Acta Mol Basis Dis. 2017;1863:2862–2870.

13.

Bouzek

Abou Alaiwa

Adam

, et al. Early lung disease exhibits bacteria-dependent and -independent abnormalities in cystic fibrosis pigs. Am J Respir Crit Care Med. 2021;204:692–702.

14.

Braux

Jourdain

Guillaume

, et al. CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth. J Cyst Fibros. 2020;19:466–475.

15.

Byrne

Stalboerger

, et al. Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation. Transplantation. 2011;91:287–292.

16.

Carrel

The transplantation of organs: a preliminary communication. 1905 [classical article]. Yale J Biol Med. 2001;74:239–241.

17.

Chang

Pezzulo

Meyerholz

, et al. Sinus hypoplasia precedes sinus infection in a porcine model of cystic fibrosis. Laryngoscope. 2012;122:1898–1905.

18.

Chen

, et al. CRISPR/Cas9-mediated knockin of human factor IX into swine factor IX locus effectively alleviates bleeding in hemophilia B pigs. Haematologica. 2021;106:829–837.

19.

Clop

Amills

Noguera

, et al. Estimating the frequency of Asian cytochrome B haplotypes in standard European and local Spanish pig breeds. Genet Sel Evol. 2004;36:97–104.

20.

Cook

Adam

Zarei

, et al. CF airway smooth muscle transcriptome reveals a role for PYK2. JCI Insight. 2017;2:e95332.

21.

Cook

Rector

Bouzek

, et al. Cystic fibrosis transmembrane conductance regulator in sarcoplasmic reticulum of airway smooth muscle. implications for airway contractility. Am J Respir Crit Care Med. 2016;193:417–426.

22.

Cooney

Abou Alaiwa

Shah

, et al. Lentiviral-mediated phenotypic correction of cystic fibrosis pigs. JCI Insight. 2016;1:e88730.

23.

Cooney

Singh

Loza

, et al. Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus. Nucleic Acids Res. 2018;46:9591–9600.

24.

Cooper

DKC

Hara

Iwase

, et al. Justification of specific genetic modifications in pigs for clinical organ xenotransplantation. Xenotransplantation. 2019;26:e12516.

25.

Cooper

DKC

Satyananda

Ekser

, et al. Progress in pig-to-non-human primate transplantation models (1998-2013): a comprehensive review of the literature. Xenotransplantation. 2014;21:397–419.

26.

Cramer

Lee

Butt

, et al. Neurologic medical device overview for pathologists. Toxicol Pathol. 2019;47:250–263.

27.

Dalmasso

Vercellotti

Platt

, et al. Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection. Transplantation. 1991;52:530–533.

28.

Diwakar

Adam

Michalski

, et al. Sonographic evidence of abnormal tracheal cartilage ring structure in cystic fibrosis. Laryngoscope. 2015;125:2398–2404.

29.

Donovan

Leidinger

McQuillen

, et al. Allograft Inflammatory factor 1 as an immunohistochemical marker for macrophages in multiple tissues and laboratory animal species. Comp Med. 2018;68:341–348.

30.

Ezashi

Telugu

Alexenko

, et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A. 2009;106:10993–10998.

31.

Food & Drug Administration. General considerations for animal studies intended to evaluate medical devices—guidance for industry and FDA staff. Published 2023. Accessed December 20, 2023. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/general-considerations-animal-studies-intended-evaluate-medical-devices#:~:text=This%20guidance%20provides%20recommendations%20for,study%20monitoring%2C%20and%20study%20evaluation.

32.

Frantz

LAF

Haile

Lin

, et al. Ancient pigs reveal a near-complete genomic turnover following their introduction to Europe. Proc Natl Acad Sci U S A. 2019;116:17231–17238.

33.

Friedemann

Mehta

Jessen

, et al. Introduction to currently applied device pathology. Toxicol Pathol. 2019;47:221–234.

34.

Gad

Schuh

JCL

. Toxicologic pathology forum opinion paper: considerations for toxicologic pathologists evaluating the safety of biomaterials and finished medical devices. Toxicol Pathol. 2018;46:366–371.

35.

Galili

Anaraki

Thall

, et al. One percent of human circulating B lymphocytes are capable of producing the natural anti-Gal antibody. Blood. 1993;82:2485–2493.

36.

Galili

Mandrell

Hamadeh

, et al. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun. 1988;56:1730–1737.

37.

Goerlich

Griffith

Hanna

, et al. The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors. J Thorac Cardiovasc Surg. 2023;165:e69–e81.

38.

Griffith

Goerlich

Singh

, et al. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med. 2022;387:35–44.

39.

Guo

Stoltz

Zhu

, et al. Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets. J Cyst Fibros. 2014;13:251–259.

40.

Gutierrez

Dicks

Glanzner

, et al. Efficacy of the porcine species in biomedical research. Front Genet. 2015;6:293.

41.

Hall

Limaye

Kulkarni

AB.

Overview: generation of gene knockout mice. Curr Protoc Cell Biol. 2009; 44:19.12.1–19.12.17.

42.

Hammer

Thein

Physiological aspects of xenotransplantation, 2001. Xenotransplantation. 2002;9:303–305.

43.

Harrison

Merrill

Murray

JE.

Renal homotransplantation in identical twins. Surg Forum. 1956;6:432–436.

44.

Heckl-Ostreicher

Binder

Kirschfink

Functional activity of the membrane-associated complement inhibitor CD59 in a pig-to-human in vitro model for hyperacute xenograft rejection. Clin Exp Immunol. 1995;102:589–595.

45.

Helke

Meyerholz

Beck

, et al. Research relevant background lesions and conditions: ferrets, dogs, swine, sheep, and goats. ILAR J. 2021;62:133–168.

46.

Helke

Swindle

MM.

Animal models of toxicology testing: the role of pigs. Expert Opin Drug Metab Toxicol. 2013;9:127–139.

47.

Helke

Wolfe

Smith

, et al. Mulberry heart disease and hepatosis dietetica in farm pigs (Sus scrofa domesticus) in a research setting. Comp Med. 2020;70:376–383.

48.

Hidiroglou

Cave

Atwall

, et al. Comparative vitamin E requirements and metabolism in livestock. Ann Rech Vet. 1992;23:337–359.

49.

Hoegger

Fischer

McMenimen

, et al. Impaired mucus detachment disrupts mucociliary transport in a piglet model of cystic fibrosis. Science. 2014;345:818–822.

50.

Hui

TCH

Brace

Hinshaw

, et al. Microwave ablation of the liver in a live porcine model: the impact of power, time and total energy on ablation zone size and shape. Int J Hyperthermia. 2020;37:668–676.

51.

Iourov

Vorsanova

Yurov

YB.

Pathway-based classification of genetic diseases. Mol Cytogenet. 2019;12:4.

52.

Iwase

Ekser

Hara

, et al. Regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition. Xenotransplantation. 2014;21:72–83.

53.

Keating

Melidone

Garcia-Polite

Preclinical evaluation of mesh implants: the pathologist’s perspective. Toxicol Pathol. 2019;47:379–389.

54.

Keele

KD.

Three early masters of experimental medicine-Erasistratus, Galen and Leonardo da Vinci. Proc R Soc Med. 1961;54:577–588.

55.

Khanna

Moutal

White

, et al. Assessment of nociception and related quality-of-life measures in a porcine model of neurofibromatosis type 1. Pain. 2019;160:2473–2486.

56.

Kirchhof

What is “preclinical device pathology”: an introduction of the unfamiliar. Toxicol Pathol. 2019;47:205–212.

57.

Klymiuk

Blutke

Graf

, et al. Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Hum Mol Genet. 2013;22:4368–4382.

58.

Lai

Kolber-Simonds

Park

, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002;295:1089–1092.

59.

Lambrigts

Sachs

Cooper

DK.

Discordant organ xenotransplantation in primates: world experience and current status. Transplantation. 1998;66:547–561.

60.

Larsen

Rivera-Marrero

Ernst

, et al. Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA. J Biol Chem. 1990;265:7055–7061.

61.

Lee

Clarke

Hoverson

, et al. MRI-guided laser interstitial thermal therapy using the Visualase system and Navigus frameless stereotaxy in an infant: technical case report. J Neurosurg Pediatr. 2021;28:50–53.

62.

Lentine

Smith

Miller

, et al. OPTN/SRTR 2021 annual data report: kidney. Am J Transplant. 2023;23:S21–S120.

63.

Ganta

Fong

Altered ion transport by thyroid epithelia from CFTR(-/-) pigs suggests mechanisms for hypothyroidism in cystic fibrosis. Exp Physiol. 2010;95:1132–1144.

64.

Tang

Vargas Buonfiglio

, et al. Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense. Am J Physiol Lung Cell Mol Physiol. 2016;310:L670–169.

65.

Villacreses

Thornell

, et al. V-type ATPase mediates airway surface liquid acidification in pig small airway epithelial cells. Am J Respir Cell Mol Biol. 2021;65:146–156.

66.

Love

Hanna

Thomas

, et al. Preclinical evaluation of a third-generation absorbable antibacterial envelope. Heart Rhythm. 2023;20:737–743.

67.

Loveland

Milland

Kyriakou

, et al. Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons. Xenotransplantation. 2004;11:171–183.

68.

Lunney

Van Goor

Walker

, et al. Importance of the pig as a human biomedical model. Sci Transl Med. 2021;13:eabd5758.

69.

Macher

Galili

The Galalpha1,3Galbeta1,4GlcNAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance. Biochim Biophys Acta. 2008;1780:75–88.

70.

Martens

Reyes

, et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation. 2017;101:e86–e92.

71.

Meyerholz

DK.

Commotio cordis and one medicine. Vet Pathol. 2023;60:291–293.

72.

Meyerholz

DK.

Lessons learned from the cystic fibrosis pig. Theriogenology. 2016;86:427–432.

73.

Meyerholz

Beck

Goeken

, et al. Glycogen depletion can increase the specificity of mucin detection in airway tissues. BMC Res Notes. 2018;11:763.

74.

Meyerholz

Lambertz

Reznikov

, et al. Immunohistochemical detection of markers for translational studies of lung disease in pigs and humans. Toxicol Pathol. 2016;44:434–441.

75.

Meyerholz

Ofori-Amanfo

Leidinger

, et al. Immunohistochemical markers for prospective studies in neurofibromatosis-1 porcine models. J Histochem Cytochem. 2017;65:607–618.

76.

Meyerholz

Reznikov

LR.

Simple and reproducible approaches for the collection of select porcine ganglia. J Neurosci Methods. 2017;289:93–98.

77.

Meyerholz

Stoltz

Gansemer

, et al. Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs. Lab Invest. 2018;98:825–838.

78.

Meyerholz

Stoltz

Namati

, et al. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Respir Crit Care Med. 2010;182:1251–1261.

79.

Meyerholz

Stoltz

Pezzulo

, et al. Pathology of gastrointestinal organs in a porcine model of cystic fibrosis. Am J Pathol. 2010;176:1377–1389.

80.

Min

Pan

Wang

, et al. Biological characteristics of captive Chinese Wuzhishan minipigs (Sus scrofa). Int Sch Res Notices. 2014;2014:761257.

81.

Miwa

Kobayashi

Nagasaka

, et al. Are N-glycolylneuraminic acid (Hanganutziu-Deicher) antigens important in pig-to-human xenotransplantation? Xenotransplantation. 2004;11:247–253.

82.

Montgomery

Stern

Lonze

, et al. Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med. 2022;386:1889–1898.

83.

Moretti

Fonteyne

Giesert

, et al. Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nat Med. 2020;26:207–214.

84.

Nakazawa

Finn

Ladich

, et al. Drug-eluting stent safety: findings from preclinical studies. Expert Rev Cardiovasc Ther. 2008;6:1379–1391.

85.

National Academies of Sciences, Engineering, and Medicine. Necessity, Use, and Care of Laboratory Dogs at the U.S. Department of Veterans Affairs. Washington, DC: National Academies Press; 2020.

86.

Nielsen

Wolstrup

Schirmer

, et al. Mulberry heart disease in young pigs without vitamin E and selenium deficiency. Vet Rec. 1989;124:535–537.

87.

Nunoya

Shibuya

Saitoh

, et al. Use of miniature pig for biomedical research, with reference to toxicologic studies. J Toxicol Pathol. 2007;20:125–132.

88.

O’Brien

Schuh

JCL

Wancket

, et al. Scientific and regulatory policy committee points to consider for medical device implant site evaluation in nonclinical studies. Toxicol Pathol. 2022;50:512–530.

89.

Ostedgaard

Meyerholz

Chen

, et al. The DeltaF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs. Sci Transl Med. 2011;3:74ra24.

90.

Ostedgaard

Moninger

McMenimen

, et al. Gel-forming mucins form distinct morphologic structures in airways. Proc Natl Acad Sci U S A. 2017;114:6842–6847.

91.

Ostedgaard

Price

Whitworth

, et al. Lack of airway submucosal glands impairs respiratory host defenses. Elife. 2020;9:e59653.

92.

Pallares

Yaeger

Janke

, et al. Vitamin E and selenium concentrations in livers of pigs diagnosed with mulberry heart disease. J Vet Diagn Invest. 2002;14:412–414.

93.

Perkins

LEL

. Preclinical models of restenosis and their application in the evaluation of drug-eluting stent systems. Vet Pathol. 2010;47:58–76.

94.

Perkins

LEL

Rippy

MK.

Balloons and stents and scaffolds: preclinical evaluation of interventional devices for occlusive arterial disease. Toxicol Pathol. 2019;47:297–310.

95.

Perleberg

Kind

Schnieke

Genetically engineered pigs as models for human disease. Dis Model Mech. 2018;11:dmm030783.

96.

Pezzulo

Tang

Hoegger

, et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature. 2012;487:109–113.

97.

Phelps

Koike

Vaught

, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;299:411–414.

98.

Pierucci-Alves

Akoyev

Stewart

III , et al. Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth. Biol Reprod. 2011;85:442–451.

99.

Porrett

Orandi

Kumar

, et al. First clinical-grade porcine kidney xenotransplant using a human decedent model. Am J Transplant. 2022;22:1037–1053.

100.

Reemtsma

McCracken

Schlegel

, et al. Renal heterotransplantation in man. Ann Surg. 1964;160:384–410.

101.

Reznikov

Dong

Chen

, et al. CFTR-deficient pigs display peripheral nervous system defects at birth. Proc Natl Acad Sci U S A. 2013;110:3083–3088.

102.

Rogan

Reznikov

Pezzulo

, et al. Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth. Proc Natl Acad Sci U S A. 2010;107:20571–20575.

103.

Rogers

Abraham

Brogden

, et al. The porcine lung as a potential model for cystic fibrosis. Am J Physiol Lung Cell Mol Physiol. 2008;295:L240–L163.

104.

Rogers

Stoltz

Meyerholz

, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science. 2008;321:1837–1841.

105.

Rousselle

Wicks

JR.

Preparation of medical devices for evaluation. Toxicol Pathol. 2008;36:81–84.

106.

Rousselle

Wicks

Tabb

, et al. Histology strategies for medical implants and interventional device studies. Toxicol Pathol. 2019;47:235–249.

107.

Sachs

DH.

The pig as a potential xenograft donor. Vet Immunol Immunopathol. 1994;43:185–191.

108.

Sakakura

Ladich

Edelman

, et al. Methodological standardization for the pre-clinical evaluation of renal sympathetic denervation. JACC Cardiovasc Interv. 2014;7:1184–1193.

109.

Sato

Kagoshima

Saitoh

, et al. Generation of alpha-1,3-galactosyltransferase-deficient porcine embryonic fibroblasts by CRISPR/Cas9-mediated knock-in of a small mutated sequence and a targeted toxin-based selection system. Reprod Domest Anim. 2015;50:872–880.

110.

Schelstraete

Clerck

Govaert

, et al. Characterization of porcine hepatic and intestinal drug metabolizing CYP450: comparison with human orthologues from A quantitative, activity and selectivity perspective. Sci Rep. 2019;9:9233.

111.

Schoen

Hirsch

Bianco

, et al. Onset and progression of calcification in porcine aortic bioprosthetic valves implanted as orthotopic mitral valve replacements in juvenile sheep. J Thorac Cardiovasc Surg. 1994;108:880–887.

112.

Schomberg

Tellez

Meudt

, et al. Miniature swine for preclinical modeling of complexities of human disease for translational scientific discovery and accelerated development of therapies and medical devices. Toxicol Pathol. 2016;44:299–314.

113.

Scriver

Waters

PJ.

Monogenic traits are not simple: lessons from phenylketonuria. Trends Genet. 1999;15:267–272.

114.

Sebek

Kramer

Rocha

, et al. Bronchoscopically delivered microwave ablation in an in vivo porcine lung model. ERJ Open Res. 2020;6:00146-2020.

115.

Shah

Meyerholz

Tang

, et al. Airway acidification initiates host defense abnormalities in cystic fibrosis mice. Science. 2016;351:503–507.

116.

Shatokhin

KS.

Problems of mini-pig breeding. Vavilovskii Zhurnal Genet Selektsii. 2021;25:284–291.

117.

Shen

Thomas

Ensley

, et al. Vitamin E and selenium levels are within normal range in pigs diagnosed with mulberry heart disease and evidence for viral involvement in the syndrome is lacking. Transbound Emerg Dis. 2011;58:483–491.

118.

Sieren

Meyerholz

Wang

, et al. Development and translational imaging of a TP53 porcine tumorigenesis model. J Clin Invest. 2014;124:4052–4066.

119.

Sieren

Quelle

Meyerholz

, et al. Porcine cancer models for translational oncology. Mol Cell Oncol. 2014;1:e969626.

120.

Sivertsen

Vie

Bernhoft

, et al. Vitamin E and selenium plasma concentrations in weanling pigs under field conditions in Norwegian pig herds. Acta Vet Scand. 2007;49:1.

121.

Spangler

Katzman

SA.

Pathological safety assessment in preclinical neurothrombectomy studies. Toxicol Pathol. 2019;47:264–279.

122.

Stanley

JRL

Keating

San Souci

. An overview on the considerations for the planning of nonclinical necropsies for medical device studies. Toxicol Pathol. 2019;47:213–220.

123.

Starzl

Marchioro

Peters

, et al. Renal heterotransplantation from baboon to man: experience with 6 cases. Transplantation. 1964;2:752–776.

124.

Stewart

Haines

Miklavcic

, et al. Safety and chronic lesion characterization of pulsed field ablation in a Porcine model. J Cardiovasc Electrophysiol. 2021;32:958–969.

125.

Stirm

Fonteyne

Shashikadze

, et al. Pig models for Duchenne muscular dystrophy—from disease mechanisms to validation of new diagnostic and therapeutic concepts. Neuromuscul Disord. 2022;32:543–556.

126.

Stoffregen

Rousselle

Rippy

MK.

Pathology approaches to determine safety and efficacy of cardiac ablation catheters. Toxicol Pathol. 2019;47:311–328.

127.

Stoltz

Meyerholz

Pezzulo

, et al. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Sci Transl Med. 2010;2:29ra31.

128.

Stoltz

Meyerholz

Welsh

MJ.

Origins of cystic fibrosis lung disease. N Engl J Med. 2015;372:351–362.

129.

Stoltz

Rokhlina

Ernst

, et al. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs. J Clin Invest. 2013;123:2685–2693.

130.

Swindle

Makin

Herron

, et al. Swine as models in biomedical research and toxicology testing. Vet Pathol. 2012;49:344–356.

131.

Tamiyakul

Kemter

Kosters

, et al. Progressive proteome changes in the myocardium of a pig model for Duchenne muscular dystrophy. iScience. 2020;23:101516.

132.

Tang

Mayersohn

Porcine prediction of pharmacokinetic parameters in people: a pig in a poke?

Drug Metab Dispos. 2018;46:1712–1724.

133.

Tellez

Dillon

Rousselle

SD.

Comprehensive preclinical postmortem evaluation of valvular prosthesis. Toxicol Pathol. 2017;45:1077–1090.

134.

Thurman

Villacreses

, et al. A single-cell atlas of large and small airways at birth in a porcine model of cystic fibrosis. Am J Respir Cell Mol Biol. 2022;66:612–622.

135.

Giriyappa

Meyerholz

, et al. Pancreatic and biliary secretion are both altered in cystic fibrosis pigs. Am J Physiol Gastrointest Liver Physiol. 2012;303:G961–G198.

136.

Olivier

Griffin

, et al. Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass. Clin Sci (Lond). 2015;128:131–142.

137.

Uthoff

Larson

Sato

, et al. Longitudinal phenotype development in a minipig model of neurofibromatosis type 1. Sci Rep. 2020;10:5046.

138.

Vahl

Grogan

Cheng

, et al. Experimental evaluation of a novel percutaneous transseptal catheter-based mitral valve replacement technology. Circ Cardiovasc Interv. 2019;12:e008002.

139.

Vatterott

Eggen

Hilpisch

, et al. Implant, performance, and retrieval of an atrial leadless pacemaker in sheep. Heart Rhythm. 2021;18:288–296.

140.

Veit

Avramescu

Chiang

, et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell. 2016;27:424–433.

141.

Wancket

LM.

Animal models for evaluation of bone implants and devices: comparative bone structure and common model uses. Vet Pathol. 2015;52:842–850.

142.

Wang

Huang

, et al. Genetic characteristics of inbred Wuzhishan miniature pigs, a native Chinese breed. J Reprod Dev. 2006;52:639–643.

143.

Wei

Zhang

, et al. Application of the transgenic pig model in biomedical research: a review. Front Cell Dev Biol. 2022;10:1031812.

144.

Weija

Dekuan

Why the character for “family” has a pig inside a house. China Daily. October 7, 2011. https://usa.chinadaily.com.cn/weekly/2011-10/07/content_13843121.htm#:~:text=One%20way%20of%20explaining%20it,a%20house%20with%20a%20pig

145.

White

Swier

Cain

, et al. A porcine model of neurofibromatosis type 1 that mimics the human disease. JCI Insight. 2018;3:e120402.

146.

Wilmut

Bai

Taylor

Somatic cell nuclear transfer: origins, the present position and future opportunities. Philos Trans R Soc Lond B Biol Sci. 2015;370:20140366.

147.

Wilson

LG.

Erasistratus, Galen, and the pneuma. Bull Hist Med. 1959;33:293–314.

148.

Xie

Tang

, et al. Acidic submucosal gland pH and elevated protein concentration produce abnormal cystic fibrosis mucus. Dev Cell. 2020;54:488–500.e5.

149.

Yamamoto

Iwase

Patel

, et al. Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout). Sci Rep. 2020;10:9771.

150.

Yang

Genome editing of pigs for agriculture and biomedicine. Front Genet. 2018;9:360.

151.

Zarei

Meyerholz

Stoltz

DA.

Early intrahepatic duct defects in a cystic fibrosis porcine model. Physiol Rep. 2021;9:e14978.

152.

Zarei

Stroik

Gansemer

, et al. Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model. Lab Invest. 2020;100:1388–1399.

153.

Zhang

Huang

Wang

, et al. Development and genome sequencing of a laboratory-inbred miniature pig facilitates study of human diabetic disease. iScience. 2019;19:162–176.

154.

Zilla

Bezuidenhout

Human

Prosthetic vascular grafts: wrong models, wrong questions and no healing. Biomaterials. 2007;28:5009–5027.

155.

Zimmerman

Karriker

Ramirez

, et al. Diseases of Swine. 11th ed. Hoboken, NJ: Wiley-Blackwell/American Association of Swine Veterinarians; 2019.

156.

Zou

Ouyang

Pang

, et al. Pathological alterations in the gastrointestinal tract of a porcine model of DMD. Cell Biosci. 2021;11:131.

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