Large Animal Model Research Articles

Myocardial blood flow quantification with SPECT

IntroductionThe addition of absolute myocardial blood flow (MBF) data improves the diagnostic and prognostic accuracy of relative perfusion imaging with nuclear medicine. Cardiac-specific gamma cameras allow measurement of MBF with SPECT. MethodsThis paper reviews the evidence supporting the use of SPECT to measure myocardial blood flow (MBF). Studies have evaluated SPECT MBF in large animal models and compared it in humans with invasive angiographic measurements and against the clinical standard of PET MBF. The repeatability of SPECT MBF has been determined in both single-site and multi-center trials. ResultsSPECT MBF has excellent correlation with microspheres in an animal model, with the number of stenoses and fractional flow reserve, and with PET-derived MBF. The inter-user coefficient of variability is ∼20% while the COV of test-retest MBF is ∼30%. SPECT MBF improves the sensitivity and specificity of the detection of multi-vessel disease over relative perfusion imaging and provides incremental value in predicting adverse cardiac events. ConclusionSPECT MBF is a promising technique for providing clinically valuable information in the assessment of coronary artery disease.

Animal Models for Investigating Osseointegration: An Overview of Implant Research over the Last Three Decades.

Dental implants and bone augmentation are among dentistry's most prevalent surgical treatments; hence, many dental implant surfaces and bone grafts have been researched to improve bone response. Such new materials were radiologically, histologically, and histomorphometrically evaluated on animals before being used on humans. As a result, several studies used animals to evaluate novel implant technologies, biocompatibility, surgical techniques, and osseointegration strategies, as preclinical research on animal models is essential to evaluate bioactive principles (on cells, compounds, and implants) that can act through multiple mechanisms and to predict animal behavior, which is difficult to predict from in vitro studies alone. In this study, we critically reviewed all research on different animal models investigating the osseointegration degree of new implant surfaces, reporting different species used in the osseointegration research over the last 30 years. Moreover, this is the first study to summarize reviews on the main animal models used in the translational research of osseointegration, including the advantages and limitations of each model and determining the ideal location for investigating osseointegration in small and large animal models. Overall, each model has advantages and disadvantages; hence, animal selection should be based on the cost of acquisition, animal care, acceptability to society, availability, tolerance to captivity, and housing convenience. Among small animal models, rabbits are an ideal model for biological observations around implants, and it is worth noting that osseointegration was discovered in the rabbit model and successfully applied to humans.

Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project

The value of pig as “large animal model” is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine–histidine–purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

Realtime assessment of vascular occlusion and reperfusion in animal models of intraoperative imaging- a pilot study.

Intraoperative monitoring of blood flow (BF) remains vital to guiding surgical decisions. Here, we report the use of SurgeON™ Blood Flow Monitor (BFM), a prototype system that attaches to surgical microscopes and implements laser speckle contrast imaging (LSCI) to noninvasively obtain and present vascular BF information in real-time within the microscope's eyepiece. The ability of SurgeON BFM to monitor BF status during reversible vascular occlusion procedures was investigated in two large animal models: occlusion of saphenous veins in six NZW rabbit hindlimbs and clipping of middle cerebral artery (MCA) branches in four Dorset sheep brain hemispheres. SurgeON BFM acquired, presented, and stored LSCI-based blood flow velocity index (BFVi) data and performed indocyanine green video angiography (ICG-VA) for corroboration. Stored BFVi data were analyzed for each phase: pre-occlusion (baseline), with the vessel occluded (occlusion), and after reversal of occlusion (re-perfusion). In saphenous veins, BFVi relative to baseline reduced to 5.2±3.7 % during occlusion and returned to 102.9±14.9 % during re-perfusion. Unlike ICG-VA, SurgeON BFM was able to monitor reduced BFVi and characterize re-perfusion robustly during five serial occlusion procedures conducted 2-5 min apart on the same vessel. Across four sheep MCA vessels, BFVi reduced to 18.6±7.7 % and returned to 120.1±27.8 % of baseline during occlusion and re-perfusion phases, respectively. SurgeON BFM can noninvasively monitor vascular occlusion status and provide intuitive visualization of BF information in real-time to an operating surgeon. This technology may find application in vascular, plastic, and neurovascular surgery.

Off-the-Shelf Synthetic Biodegradable Grafts Transform In Situ into a Living Arteriovenous Fistula in a Large Animal Model.

Current vascular access options require frequent interventions. In situ tissue engineering (TE) may overcome these limitations by combining the initial success of synthetic grafts with long-term advantages of autologous vessels by using biodegradable grafts that transform into autologous vascular tissue at the site of implantation. Scaffolds (6 mm-Ø) made of supramolecular polycarbonate-bisurea (PC-BU), with a polycaprolactone (PCL) anti-kinking-coil, are implanted between the carotid artery and jugular vein in goats. A subset is bio-functionalized using bisurea-modified-Stromal cell-derived factor-1α (SDF1α) derived peptides and ePTFE grafts as controls. Grafts are explanted after 1 and 3 months, and evaluated for material degradation, tissue formation, compliance, and patency. At 3 months, the scaffold is resorbed and replaced by vascular neo-tissue, including elastin, contractile markers, and endothelial lining. No dilations, ruptures, or aneurysms are observed and grafts are successfully cannulated at termination. SDF-1α-peptide-biofunctionalization does not influence outcomes. Patency is lower in TE grafts (50%) compared to controls (100% patency), predominantly caused by intimal hyperplasia. Rapid remodeling of a synthetic, biodegradable vascular scaffold into a living, compliant arteriovenous fistula is demonstrated in a large animal model. Despite lower patency compared to ePTFE, transformation into autologous and compliant living tissue with self-healing capacity may have long-term advantages.

Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts.

Static cold storage (SCS), the current clinical gold standard for organ preservation, provides surgeons with a limited window of time between procurement and transplantation. In vascularized composite allotransplantation (VCA), this time limitation prevents many viable allografts from being designated to the best-matched recipients. Machine perfusion (MP) systems hold significant promise for extending and improving organ preservation. Most of the prior MP systems for VCA have been built and tested for large animal models. However, small animal models are beneficial for high-throughput biomolecular investigations. This study describes the design and development of a multiparametric bioreactor with a circuit customized to perfuse rat abdominal wall VCAs. To demonstrate its concept and functionality, this bioreactor system was employed in a small-scale demonstrative study in which biomolecular metrics pertaining to graft viability were evaluated non-invasively and in real time. We additionally report a low incidence of cell death from ischemic necrosis as well as minimal interstitial edema in machine perfused grafts. After up to 12 h of continuous perfusion, grafts were shown to survive transplantation and reperfusion, successfully integrating with recipient tissues and vasculature. Our multiparametric bioreactor system for rat abdominal wall VCA provides an advanced framework to test novel techniques to enhance normothermic and sub-normothermic VCA preservations in small animal models.

The effects of CO2 levels and body temperature on brain interstitial pH alterations during the induction of hypoxic-ischemic encephalopathy in newborn pigs

Brain interstitial pH (pHbrain) alterations play a crucial role in the development of hypoxic-ischemic (HI) encephalopathy (HIE) caused by asphyxia in neonates. The newborn pig is one of the most suitable large animal models for studying HIE, however, compared to rats, experimental data on pHbrain alterations during HIE induction are limited. The major objective of the present study was thus to compare pHbrain changes during HIE development induced by experimental normocapnic hypoxia (H) or asphyxia (A), elicited with ventilation of a gas mixture containing 6%O2 or 6%O2/20%CO2, respectively for 20 min, under either normothermia (NT) or hypothermia (HT) (38.5 ± 0.5 °C or 33.5 ± 0.5 °C core temperature, respectively) in anesthetized piglets yielding four groups: H-NT, A-NT, H-HT, and A-HT. pHbrain changes during HI stress and the 60 min reoxygenation period were measured using a pH-selective microelectrode inserted into the parietal cortex through an open cranial window. In all groups, the pHbrain response to HI stress was acidosis, at the nadir pHbrain values dropped from the baseline of 7.27 ± 0.02 to H-NT:5.93 ± 0.30, A-NT:5.90 ± 0.52, H-HT:6.81 ± 0.27, and A-HT:6.27 ± 0.24 indicating that (1) H and A elicited similar, severe brain acidosis under NT greatly exceeding pH changes in arterial blood (pHa dropped to 7.24 ± 0.07 and 6.78 ± 0.03 from 7.52 ± 0.06 and 7.50 ± 0.05, respectively), and (2) HT ameliorated more the brain acidosis induced by H than by A. In all four groups, pHbrain was restored to baseline values without an alkalotic overshoot during the observed reoxygenation, Our findings suggest that under NT either H or A – both commonly employed HI stresses to elicit HIE in piglet models – would result in a similar acidotic pHbrain response without an alkalotic component either during the HI stress or the early reoxygenation period.

Pharmacokinetic evaluation of a single chain antibody fragment against scorpion toxins in sheep

A key aspect during the development of antivenoms is the evaluation of the efficiency and security of the therapeutic molecules. In this work, we report the pharmacokinetic analysis of a neutralizing single chain antibody fragment named LR (scFv LR) where three sheep were used as a large animal model. The animals were injected through i.v. route with 2 mg of scFv LR. Blood samples were drawn every minute within the first 15 min, the sampling continues at 20, 25, 30, 45, 60, 90, 120 min, subsequently at 1-h intervals, 3, 4, 5, 6 h, two more samples at 9 and 12 h and, two more samples at 24 and 48 h and finally at one-day intervals during 4 days. scFv LR levels were measured from blood serum and urine samples by an ELISA. The pharmacokinetics of the experimental data was analyzed using the three-exponential kinetics. The value of the fast initial component (τ1=0.409±0.258min) indicated that the scFv is distributed rapidly into the tissues. The mean residence time, MRT, was 45 ± 0.51 min and the clearance (CL), 114.3 ± 14.3 mL/min. From urine samples it was possible to detect significant amounts of scFv LR, which is evidence of renal elimination.

Abstract 1456: First successful engraftment of human liver cancer cell line in highly robust immunocompromised porcine model to test the tumor ablation efficacy by histotripsy

Abstract Liver tumor, commonly called Hepatocellular carcinoma(HCC), is the most common type of primary liver malignancy. It is the fourth leading cause of cancer-related deaths. Late diagnosis, location of the tumor, tumor burden, and metastases makes liver tumor very challenging to treat by liver transplantation, surgical procedures, and other ablation techniques like cryoablation and thermal ablation. Also, the absence of a preclinical animal model makes it challenging to develop and explore the feasibility of treatment modalities. Histotripsy is a non-invasive, non-ionizing, non-thermal, image-guided focused ultrasound ablation treatment method that uses high-pressure pulses to create acoustic cavitation, a “bubble cloud,” at the target. The bubble cloud expands and collapses, which ablates the tissue into an acellular homogenate. Pigs are ideal animal models as they closely resemble human anatomy and are significant in improving the translation to human patients. This study aimed to establish the feasibility of successfully ablating liver tumors by histotripsy using an immunocompromised porcine model to generate HepG2 human liver cancer cell line tumors in the liver of the immunocompromised pigs. The orthotopic porcine model was established using RAG2/IL2RG double knockout immunocompromised pigs. HepG2 cells were injected orthotopically into the liver of immunocompromised pigs. Three weeks after the injections, CT images and necropsy indicated successful engraftment and growth of liver tumors in the pigs. The ultrasound images and post-histotripsy treatment histology images showed confirmed ablation zones in the liver. Therefore, in conclusion, our preliminary results in the study could demonstrate, for the first time, a highly robust model of human liver cancer in a large animal model. Soon, we plan to conduct more such studies and explore all the possible utilities of these immunocompromised porcine models to develop therapeutic strategies to increase the efficacy of liver tumor ablation by histotripsy. Citation Format: Tamalika Paul, Khan M. Imran, Jessica Gannon, Brie Trusiano, Kristin Eden, Hannah Ivester, Madeline Mott, Manali Powar, Michael Edwards, Christopher Byron, Sherrie Clark-Deener, Kiho Lee, Eli Vlaisavljevich, Irving Coy Allen. First successful engraftment of human liver cancer cell line in highly robust immunocompromised porcine model to test the tumor ablation efficacy by histotripsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1456.

Abstract 3392: Generation and characterization of transgenic oncopig model for lung cancer

Abstract Lung cancer (LC) is the leading cause of cancer-associated death in the United States. Preclinical findings in small animal models do not translate into human clinics. There is an urgent need to develop relevant preclinical large animal models for LC. We describe a novel method to induce LC in transgenic pigs carrying Cre-inducible KrasG12D and TrP53R167H mutations (Oncopigs). The Oncopigs (n=12; 9 females, and 3 males) at 9 weeks of age were anesthetized and adenovirus (1 × 1011 PFU) carrying Cre recombinase gene (Ad-Cre) ± polybrene (1:100) ± IL-8 (5 ng/ml) was injected via flexible bronchoscopy with two techniques: (1) Bronchial lavage (n=6) and (2) Transbronchial needle injection (n=6) into different lung lobes. As controls, a combination of polybrene ± IL-8 was injected without AdCre into the contralateral lobes in the same pigs (n=8). Oncopigs were monitored via clinical monitoring, blood counts, basic metabolic panel, and contrast-enhanced computed tomography (CT) imaging for LC progression and metastasis at 3-, 4-, 7-, 10-, 16-, and 26 weeks post-intervention. All animals tolerated the injections with expected weight gains. Blood counts and metabolic panels remained normal during LC progression. At the site of injection/lavage, lung masses were detected on CT imaging at 3 weeks post-injections. However, a decrease in masses was observed on CT at 7 weeks post-intervention. The decrease in size of the lung masses coincided with increasing attenuation. No distant metastases were observed on CT imaging. Lavage-based lesions appeared to be more heterogeneous with surrounding inflammatory changes. Transbronchial-injection-based lesions appeared more consolidated and coin-shaped without surrounding inflammatory changes. Control injection sites did not show any mass growth on CT imaging. Immunohistochemistry analysis on lung tumors revealed increased pancytokeratin, trichrome, E-cad, Ki-67, IBA, and CD3 expression. Further, immunofluorescence revealed decreased E-Cad, CK-19, and Zo-1 expression, whereas increased expression of N-Cad, FN1, snail, CD44, α-SMA, and γ-catenin, was observed in the LC and metastatic tissue compared to normal. We successfully generated and characterized a novel transgenic Oncopig LC model. There is robust tumor production status post AdCre delivery. One of four Oncopigs had carcinomatous metastatic skin growth. The remaining 7 Oncopigs are monitored longitudinally for tumor growth and development of metastasis. Citation Format: Kirtan Joshi, Nagabhishek Sirpu, Amanda Schmelzle, Ravikanth Poonooru, Benjamin Nelson, Pradeep Subramanyam, Colleen Garrett, Mariana Sponchiado, Sarah Schlink, Samantha Gerb, Jesse Porter, Dae Kim, Jeffrey Kunin, Jeffrey Bryan, Timothy Hoffman, Bhanu Telugu, Jussuf Kaifi, Satyanarayana Rachagani. Generation and characterization of transgenic oncopig model for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3392.

Abstract 1760: Mutational landscape of the canine acute myeloid leukemia exome

Abstract Over 21,000 newly diagnosed cases of human acute myeloid leukemia (hAML) and 10,000 hAML-related deaths are reported in the United States every year. hAML is considered a highly diverse aggressive hematopoietic malignancy, with over 16 recurrent gene rearrangements, 60 recurrent point mutations, and hundreds of rarely mutated genes. There are large ongoing multi-institutional studies devoted to advancing medical management for hAML by providing targeted therapeutics to patients based on their molecular characteristics. A preclinical spontaneous large animal model for testing novel therapies could accelerate the development of better treatments in people. Dogs spontaneously develop AML with similar clinical features and outcomes. As such, the dog may be a useful pre-clinical model, but little is known about the molecular landscape and/or drivers of canine AML (cAML). The goal of this work was to identify somatic mutation candidates in cAML and determine the overlapping features with hAML. We utilized whole exome sequencing at 300x coverage to identify candidate mutations in 51 cAML samples. Somatic variant calling was performed per the GATK best practice recommendations using Mutect2 and a reference panel of normals (n=77) and a variant call file of canine germline variants. We focused our analysis on novel variants predicted to have a moderate to high impact and that occurred in genes implicated in cancer. At least one variant was identified in genes involved in the RTK/RAS pathway (e.g. NRAS, KRAS, PTPN11, FLT3, KIT) in over 75% of cAML samples, a trend that is also found in people. Additionally, recurrent point mutations were predicted in NRAS and KRAS, and some of which, such as the codon changes G13R and Q61R/H in NRAS, were noted to be shared between hAML and cAML. Mutations in DNMT3A were uncommon (n=3/44) and NPM1 mutations were not identified in our cohort, both of which are common hAML-associated genes. However, mutations in other epigenetic modifiers, such as KDM5C and KDM6A, occurred at a high frequency. This data provides insight into the candidate mutations potentially driving cAML pathogenesis and highlights the overlapping molecular features with hAML. Citation Format: Robert Harris, Evan Conaway, Anne Avery. Mutational landscape of the canine acute myeloid leukemia exome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1760.

Abstract 6336: Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination

Abstract Background: Metastatic osteosarcoma (OSA) has a dismal prognosis despite novel targeted therapies, including CAR T cells. Unleashing CAR T cell therapy against poorly immunogenic cancers requires new technologies that activate the tumor microenvironment (TME), while inducing CAR T cell migration, proliferation, and persistence into tumor to sustain tumor-specific immunity. We have developed tumor RNA-loaded nanoparticle (CD70-encoding RNA-NP) vaccines to overcome CD70-specific CAR T cell limitations in OSA. Methods: Using two murine solid tumor models (K7M2 osteosarcoma and B16F0 melanoma, in Balb/c and C57Bl/6 background) highly metastatic to lungs, we implant K7M2 and B16F0 intravenously (i.v.). We subsequently generated NPs complexed with RNA encoding for CD70 antigen and control vaccine (GFP RNA). RNA-NPs are administered intravenously beginning on day 5 post-tumor implantation and day 7, 14 & 21. Mouse CD70-specific CAR T cells are manufactured after activation of T cells with CD3 and CD28 antibodies for 2 days. CAR T cells are ready for use in downstream experiments 2 days after pMSGV8-mCAR transduction and cells were injected (i.v.) on day 6. Results: In preliminary data, we show that intravenous administration of CD70-encoding RNA-NPs rapidly recruit CD70-directed CAR T cells into tumor stroma unlocking their activity in two immunocompetent aggressive lung metastatic tumor models. Interestingly, regression of CD70-expressing tumors associates with CAR T cell mobilization out of peripheral blood and into reticuloendothelial (RES) tissues (i.e., lung, and spleen), a phenomenon that correlates with upregulation of vaccine-induced type I interferon (IFN) and activation of CCR2 pathway. In addition to the CAR T-mediated antitumor killing, an antigen spreading phenomenon was observed. The long-term survival benefit (150 days) provided by this approach reached 70-80% in both models compared with 0-10% of control groups. Conclusions: Based on these findings, we hypothesize that tumor trafficking and persistence of CD70 CAR T cells depend on coordinated cytokine/chemokine signaling that can be ignited through intravenous RNA-NP vaccination. RNA-NPs in combination with CAR T cells prolong long-term survival of mice bearing these aggressive tumor models which was not only mediated by CAR T cells but also by activation of endogenous adaptive immunity. Future studies include the evaluation of this approach in large animal models prior to translation into first-in-human clinical trials. Citation Format: Arnav Barpujari, Rui Liu, Elizabeth Ogando-Rivas, Xiaojie Ma, Haipeng Taoa, Hector Gomez, Duane Mitchell, Jianping Huang, Elias Sayour, Paul Castillo. Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6336.

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs invivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Spring-mediated distraction enterogenesis may alter the course of adaptation in porcine short bowel syndrome.

Severe forms of short bowel syndrome (SBS) resulting in chronic intestinal failure (IF) have limited therapeutic options, all of which are associated with significant morbidities. Spring-mediated distraction enterogenesis (SMDE) uses an intraluminal self-expanding spring to generate mechanical force to induce intestinal stretching and sustained axial growth, providing a promising novel approach for patients with SBS. Previous studies have established this method to be safe and effective in small and large animal models. However, SMDE has previously not been implemented in a large, clinically relevant animal model. Juvenile mini-Yucatan pigs with 75% of their small intestine resected had intraluminal springs placed after an initial adaptive period. Morphological and histological assessments were performed on SMDE segments compared to the control region of the intestine undergoing normal adaptive responses to resection. While the initial histologic adaptive response observed following resection was attenuated after a month, the SMDE segments instead augmented these adaptive changes. Specifically, intestinal length increased 2-fold in SMDE segments, and the widths of the epithelial, muscularis, and serosal layers were enhanced in SMDE compared with control segments of the same animal. This data suggests that morphologic intestinal adaptation may be enhanced with SMDE in the setting of SBS. Here we demonstrate the successful and reproducible implementation of SMDE in a large animal model in the setting of prior intestinal resection, making SMDE a viable and novel approach for SBS to be explored further.

Characterizing the immune response to myocardial infarction in pigs

Though myocardial infarction (MI) in pigs is a well-established translational large animal model, it has not yet been widely used for immunotherapy studies, and a comprehensive description of the immune response to MI in this species is lacking. We induced MI in Landrace pigs by balloon occlusion of the left anterior descending artery over 90 min. Within 14 days, the necrotic myocardium was progressively replaced by scar tissue with involvement of myofibroblasts. We characterized the immune response in the heart ex vivo by (immuno)histology, flow cytometry, and RNA sequencing of myocardial tissue on days 3, 7, and 14 after MI. Besides a clear predominance of myeloid cells among heart-infiltrating leukocytes, we detected activated T cells and an increasing proportion of CD4+ Foxp3+ regulatory T cells (Treg), especially in the infarct core—findings that closely mirror what has been observed in mice and humans after MI. Transcriptome data indicated inflammatory activity that was persistent but markedly changing in character over time and linked to extracellular matrix biology. Analysis of lymphocytes in heart-draining lymph nodes revealed significantly higher proliferation rates of T helper cell subsets, including Treg on day 7 after MI, compared to sham controls. Elevated frequencies of myeloid progenitors in the spleen suggest that it might be a site of emergency myelopoiesis after MI in pigs, as previously shown in mice. We thus provide a first description of the immune response to MI in pigs, and our results can aid future research using the species for preclinical immunotherapy studies.

Longitudinal CT-based finite element analyses provide objective fracture healing measures in an ovine tibia model.

Measuring the healing status of a bone fracture is important to determine the clinical care a patient receives. Implantable devices can directly and continuously assess the healing status of fracture fixation constructs, while subject-specific virtual biomechanical tests can noninvasively determine callus structural integrity at single time points. Despite their potential for objectification, both methods are not yet integrated into clinical practice with further evidence of their benefits required. This study correlated continuous data from an implantable sensor assessing healing status through implant load monitoring with computer tomography (CT) based longitudinal finite element (FE) simulations in a large animal model. Eight sheep were part of a previous preclinical study utilizing a tibial osteotomy model and equipped with such a sensor. Sensor signal was collected over several months, and CT scans were acquired at six interim time points. For each scan, two FE analyses were performed: a virtual torsional rigidity test of the bone and a model of the bone-implant construct with the sensor. The longitudinal simulation results were compared to the sensor data at corresponding time points and a cohort-specific empirical healing rule was employed. Healing status predicted by both in silico simulations correlated significantly with the sensor data at corresponding time points and correctly identified a delayed and a nonunion in the cohort. The methodology is readily translatable with the potential to be applied to further preclinical or clinical cohorts to find generalizable healing criteria. Virtual mechanical tests can objectively measure fracture healing progressing using longitudinal CT scans.

Natural Compounds and Biomimetic Engineering to Influence Fibroblast Behavior in Wound Healing.

Throughout history, natural products have played a significant role in wound healing. Fibroblasts, acting as primary cellular mediators in skin wound healing, exhibit behavioral responses to natural compounds that can enhance the wound healing process. Identifying bioactive natural compounds and understanding their impact on fibroblast behavior offers crucial translational opportunities in the realm of wound healing. Modern scientific techniques have enabled a detailed understanding of how naturally derived compounds modulate wound healing by influencing fibroblast behavior. Specific compounds known for their wound healing properties have been identified. Engineered biomimetic compounds replicating the natural wound microenvironment are designed to facilitate normal healing. Advanced delivery methods operating at micro- and nano-scales have been developed to effectively deliver these novel compounds through the stratum corneum. This review provides a comprehensive summary of the efficacy of natural compounds in influencing fibroblast behavior for promoting wound regeneration and repair. Additionally, it explores biomimetic engineering, where researchers draw inspiration from nature to create materials and devices mimicking physiological cues crucial for effective wound healing. The review concludes by describing novel delivery mechanisms aimed at enhancing the bioavailability of natural compounds. Innovative future strategies involve exploring fibroblast-influencing pathways, responsive biomaterials, smart dressings with real-time monitoring, and applications of stem cells. However, translating these findings to clinical settings faces challenges such as the limited validation of biomaterials in large animal models and logistical obstacles in industrial production. The integration of ancient remedies with modern approaches holds promise for achieving effective and scar-free wound healing.

Onward to Better Surgery - the Critical Need for Improved Ex Vivo Testing and Training Methods.

Guided surgery has demonstrated significant improvements in patient outcomes in some disease processes. Interest in this field has led to substantial growth in the technologies under investigation. Most likely no single technology will prove to be "best," and combinations of macro- and microscale guidance-using radiological imaging navigation, probes (activatable, perfusion, and molecular-targeted; large- and small-molecule), autofluorescence, tissue intrinsic optical properties, bioimpedance, and other characteristics-will offer patients and surgeons the greatest opportunity for high-success/low-morbidity medical interventions. Problems are arising, however, from the lack of valid testing formats; surgical training simulators suffer the same problems. Small animal models do not accurately recreate human anatomy, especially in terms of tissue volume. Large animal models are expensive and have difficulty replicating many pathological states, particularly when molecular specificity for individual cancers is required. Furthermore, the sheer number of technologies and the potential for synergistic combination leads to exponential growth of testing requirements that is unrealistic for in vivo testing. Therefore, critical need exists to expand the ex vivo/in vitro testing platforms available to investigators and, once validated, a need to increase the acceptance of these methods for funding and regulatory endpoints. Herein is a review of the available ex vivo/in vitro testing formats for guided surgery, a review of their advantages/disadvantages, and consideration for how our field may safely and more swiftly move forward through stronger adoption of these testing and validation methods.