Preclinical Large Animal Model Research Articles

Evaluation of nonbiomedical and biomedical grade alginates for the transplantation of genetically modified articular chondrocytes to cartilage defects in a large animal model in vivo

Genetically modified chondrocytes embedded in alginate improve cartilage repair in experimental models, and alginates are clinically used for articular chondrocyte transplantation. In the present study, we tested the hypothesis that the alginate system allows for sustained transgene expression in cartilage defects in a preclinical large animal model in vivo. Primary cultures of ovine articular chondrocytes were transfected with the Photinus pyralis luc or the Escherichia coli lacZ genes in monolayer culture in vitro using eight different nonviral compounds. Optimally transfected chondrocytes were encapsulated in spheres composed of nonbiomedical or biomedical grade alginates for evaluation of luciferase expression, cell numbers and viabilities in vitro. Transfected chondrocytes encapsulated in spheres comprised of the different alginates were then implanted into osteochondral defects in the knee joints of sheep to examine the profiles of transgene expression in vivo. Ovine articular chondrocytes were efficiently transfected with FuGENE 6. Transgene expression was detectable after encapsulation in the alginates over 21 days in vitro. Transplantation of genetically modified chondrocytes to cartilage defects in vivo resulted in maximal transgene expression on day 1 after transfection, with a decrease by day 21, the longest time point evaluated. Remarkably, the reduction in luciferase activity was less pronounced when biomedical grade alginates were employed, compared to nonbiomedical grade alginates, suggesting that such alginates might be better suited to support elevated transgene expression after transplantation of genetically modified chondrocytes. This approach may be of value to study the effects of potential therapeutic genes upon cartilage repair in a clinically relevant setting.

108 A CRITICAL-SIZE CRANIOFACIAL BONE DEFECT MODEL IN THE YORKSHIRE PIG

Substantial and innovative developments in the field of bone tissue engineering have prompted increased demand for suitable pre-clinical large animal models. The pig has several advantages over other non-primate species, including availability, rapid growth, large litters, and similar anatomy and physiology to humans. These characteristics make them ideal models for research in diverse applications such as cardiovascular disease, pharmacological activity testing, and organ transplantation. There has been an increased interest in the use of swine as a model for bone healing and grafting techniques. Maxillofacial surgeons strive to develop the best therapy for large bone defects in the face resulting from tumour resection, congenital abnormalities, and traumatic injuries. Creating a model to study a critical-sized bone defect in the mandible, which does not spontaneously heal without clinical intervention, would be a method to test growth factors and synthetic bone graft therapies. However, the size of bone defect required to create this condition has not been ascertained. In the current study, we examined the in vivo healing response for 4, 8, and 16 weeks of surgically created bone defects in the posterior region of the pig mandible. Yorkshire barrows (n = 12) 6–7 months of age were used for the study. All animal experiments conformed to the University of Illinois Institutional Animal Care and Use Committee (IACUC) guidelines. Animals were maintained under general anaesthesia and transcortical, circular defects with diameters of 6, 10, 16, or 25 mm were created on both sides of the mandible. The presence and amount of calcified tissue was assessed using radiographs and dual energy x-ray absorptiometry (DEXA). Tissue morphology was examined using hard-tissue histological methods and a light microscope. Defect diameters of 6, 10, and 16 mm had completed healing or were in the process of healing within the 16-week timeframe of the study. Compared to controls, average percent differences in bone mineral density, in order of increasing defect size, were 0.62%, 28.1%, and 54.5%, respectively. In contrast, 25 mm diameter defects displayed limited collagenous tissue ingrowth, and the presence of calcified tissue was not detected, as indicated by radiographs and histological staining. As the defect size increased, the time required to heal was prolonged until a critical size was determined and normal bone was not completely regenerated. In conclusion, circular defects in the posterior region of the pig mandible with diameters equal or greater than 25 mm will result in limited healing without additional medical intervention and can be termed critical-sized defects. This porcine model will allow for the rapid development and testing of new approaches for the repair of damaged bone, which is especially prevalent in the craniofacial area. This work was partially supported by the Carle Foundation Hospital (#2007-04072) and the Illinois Regenerative Medicine Institute (IDPH #63080017).

Stem Cell Therapy for Ischemic Heart Disease

Stem cell transplantation has emerged as a novel treatment option for ischemic heart disease. Different cell types have been utilized and the recent development of induced pluripotent stem cells has generated tremendous excitement in the regenerative field. Bone marrow-derived multipotent progenitor cell transplantation in preclinical large animal models of postinfarction left ventricular remodeling has demonstrated long-term functional and bioenergetic improvement. These beneficial effects are observed despite no significant engraftment of bone marrow cells in the myocardium and even lower differentiation of these cells into cardiomyocytes. It is thought to be related to the paracrine effect of these stem cells, which secrete factors that lead to long-term gene expression changes in the host myocardium, thereby promoting neovascularization, inhibiting apoptosis, and stimulating resident cardiac progenitor cells. Future studies are warranted to examine the changes in the recipient myocardium after stem cell transplantation and to investigate the signaling pathways involved in these effects.

Comparative Assessment of the Osteoconductive Properties of Different BiomaterialsIn VivoSeeded with Human or Ovine Mesenchymal Stem/Stromal Cells

Mesenchymal stromal/stem cells (MSC), when used in combination with biomaterial scaffolds, have been shown to contribute at varying efficiencies to bone and cartilage regeneration in preclinical large animal models and human clinical trials. In an orthopedic context, identification of the optimal scaffold, which is capable of inducing tissue regeneration, has been the subject of numerous studies. In the present study, we show that ex vivo-expanded MSC from human and ovine bone marrow display similar phenotypic properties, but exhibit differences in their ability to form bone in vivo when transplanted with different biocompatible scaffold composites. We found that the ovine MSC formed ectopic bone on all scaffolds tested with the exception of collagen-based demineralized bone matrix. In contrast, human MSC in general formed less bone and only on those biomaterials composed of ceramic particles containing at least 15% hydroxyapatite. This study demonstrates the differences in bone formation potential between human and ovine MSC in vivo based on the osteoconductive properties of different bioscaffolds currently being used for orthopedic clinical applications.

Hepatic artery vs. portal vein infusion of microbeads: a large animal pre-clinical model evaluating the intrahepatic capacity for cell infusion and imaging

Islet xeno-transplantation via the portal vein has been proposed as an alternative of islet allo-transplantation for treatment of type 1 diabetes. However, the precise hepatic capacity has not been addressed. We mimicked cell transplantation by infusion of dogs with alginate/poly-1-lysine/alginate (APA) microbeads via either the portal vein (PV) or hepatic artery (HA). The maximal adaptable capacity for infused microbeads was evaluated by examination of vasculature, microvasculature, hepatic hemodynamics, portal vein, and hepatic artery pressures and liver function in the microbead recipient dogs. PV but not HA dogs demonstrated elevated portal pressure during the infusion procedure in a dose-dependent manner. Four out of twelve PV dogs infused with 32,000 microbeads/kg developed acute liver infarction within 24 h after infusion with four of the remaining eight animals developing portal venous thrombosis within 24 h following infusion. All PV animals demonstrated abnormal alanine aminotransferase (ALT) values, and the extent and duration of increased ALT levels correlated with the increase in the number of microbeads infused. In contrast, HA animals infused with as many as 32,000 microbeads/kg had neither portal thrombosis nor abnormal liver function. The capacity for intrahepatic cell infusion is finite and the intrahepatic artery may have a less hemodynamic interference impact on transplantation of cells into the liver when a larger volume of cells is required to achieve curable outcomes in both allo- and xeno-transplantation.

Monitoring of liver cell transplantation by MRI

Aim: Liver cell transplantation (LCT) is a promising approach for treating certain liver diseases. However, monitoring of LCT is challenging. Labelling with micron-sized iron oxide particles (MPIO) enables detection of cells by magnetic resonance imaging (MRI). Aim of this study was to investigate the feasibility of non-invasive monitoring of LCT by MRI in a preclinical large animal model.

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.

Novel Ovine Model of Heart Failure Induced by Autologous Platelet Aggregates

Introduction: There are several animal models using coronary occlusion that try to mimic ischemic heart failure (IHF). These techniques create infarctions by mechanical disruption of blood flow but lack the biomass of thrombus that occurs during plaque rupture that we see clinically. We propose a novel pre-clinical large animal model of cardiac failure developed by direct injections of autologous platelet aggregates (APAs) into the coronary circulation which results in IHF. Methods: Platelets were isolated from the whole ovine blood before embolization procedure (EP) and aggregated using thrombin.

Efficacy testing of topical delivered moxifloxacin against MRSA in a porcine wound infection model

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of infections in wounds and associated with high morbidity [1]. The emergence of bacterial resistance to multiple antibiotics [2] has led to an increased urgency to develop new therapeutic options for wound infections. The aim of this study was to assess the antimicrobial activity of moxifloxacin in a preclinical large animal model of MRSA wound infection. Methods: Six full thickness BO-wound chambers [3] were implanted on each flank of 2 female Goettingen minipigs under general anaesthesia. 7 days after implantation wounds were inoculated with 1 × 108 colony forming units (cfu) of MRSA. After wound infection had been established and quantified (3 days after inoculation), wounds were randomised and topically treated with either placebo-gel (carrier-control n = 7), 0.1 % linezolid (n = 3), 0.1 % mupirocin (n = 3), 2 % mupirocin (commercial ointment; n = 4) or 0.1 % moxifloxacin (n = 7). Topical treatment and wound fluid collection was carried out every other day for 2 weeks for quantitative bacterial analysis to monitor wound infection. Animals were photographed every day to document macroscopic signs of infection. The animals were sacrificed 21 days post-inoculation and tissue biopsies were taken for histological analysis and quantitative bacterial counts. Results: No macroscopic difference was seen between the active treatment groups, whereas pus accumulation was seen in the carrier control group. Pre-treatment all wounds showed a high and stable bacterial infection (> 105 cfu/ml wound fluid). Antibiotic therapy with linezolid, mupirocin and moxifloxacin significantly reduced bacterial counts in wound fluid and tissue in contrast to the carrier control. Moxifloxacin showed a strong antimicrobial activity (with up to 103 reduction in bacterial counts) compared to carrier control (fluid p = 0.012, tissue p = 0.013). Linezolid and mupirocin showed comparable results. Conclusion: All antibiotics showed high efficacy towards MRSA infection in this porcine full-thickness wound infection model. The activity of moxifloxacin is comparable to that of clinically used antibiotics (linezolid, mupirocin) and no side effects were observed. Moxifloxacin has promise to be a potential new therapeutic option for the treatment of MRSA wound infection.

Tissue-Engineered Small Intestine and Stomach Form from Autologous Tissue in a Preclinical Large Animal Model

Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche. A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

Induction of Tolerance to an Allogeneic Skin Flap Transplant in a Preclinical Large Animal Model

Clinical composite tissue allotransplantation can adequately reconstruct defects that are not possible by other means. However, immunosuppressant toxicity limits the use of these techniques. Clinical attempts to reduce the amount of immunosuppression required by induction of an immunologically permissive state have so far been unsuccessful. The aim of this study was to induce tolerance in a preclinical large animal model. Donor hematopoietic stem cell (HSC) engraftment was induced by T-cell depletion, irradiation, and a short course of cyclosporine administered to the recipient, along with a hematopoietic cell infusion from a single haplotype major histocompatibility complex (MHC) mismatched donor. Skin was then allotransplanted from the donor. Both primarily vascularized skin flaps and secondarily vascularized conventional skin grafts were allotransplanted to investigate if the mode of transplantation affected outcome. Control animals received the skin allotransplants without conditioning. Tolerance was defined as no evidence of rejection at 90 days following transplantation. Conventional skin grafts only achieved prolonged survival (<65 days) in HSC-engrafted animals ( P < .01). In contrast, there was indefinite skin flap survival with the achievement of tolerance in HSC-engrafted animals; this was confirmed on histology with donor-specific unresponsiveness on MLR and CML. Furthermore, a conventional skin donor graft subsequently applied to an animal tolerant to a skin flap was not rejected and did not trigger skin flap rejection. To our knowledge, this is the first time skin tolerance has been achieved across a MHC barrier in a large animal model. This is a significant step toward the goal of clinical skin tolerance induction.

The Cornucopia of “Pleiotropic” Actions of Statins

See related article, pages 255–264 Initially described in patients with coronary artery disease undergoing coronary artery bypass graft (CABG) surgery, myocardial “hibernation” denotes a state of adaptive reduction in myocardial contractile function in response to limited energy supply caused by reduced blood flow.1,2 In this precarious equilibrium, the myocardium can remain viable for sustained periods of time (although not indefinitely) and slowly resumes contractile activity on restoration of perfusion.2,3 Hibernating myocardium resulting from a flow-limiting coronary artery stenosis is a common clinical entity that contributes to myocardial dysfunction in many patients with ischemic heart disease1,2; its clinical importance stems from the fact that is it is potentially reversible. The pathophysiological basis of this syndrome was proposed as a conceptual framework in 19924 and subsequently tested in various animal models5–8: it is likely to involve recurrent brief episodes of ischemia, which initially result in repetitive “stunning” with normal resting flow and eventually cause a persistent decrease in resting function and flow.2,4–6,8 Over time, these changes lead to myocyte loss resulting from degenerative changes and apoptosis.2,8 Because mechanical dysfunction is caused by a restriction in blood flow, revascularization has traditionally been the cornerstone of therapy for myocardial hibernation. Indeed, restoration of blood flow via CABG or percutaneous coronary interventions often improves the mechanical performance of hibernating myocardium.1–3,8,9 In those cases in which these procedures are not feasible, however, there is a need for alternative approaches. Direct transmyocardial laser revascularization10 and intramyocardial gene transfer of phVEGF16511 or fibroblast growth factor-57 have been reported to enhance perfusion and contractile reserve and improve regional function in experimental models of hibernation. Transplantation of circulating progenitor cells following revascularization has also been reported to attenuate hibernation (ie, to increase coronary flow reserve …

Human beta‐defensin‐3 promotes wound healing in infected diabetic wounds

Infected wounds present a major complication in patients with diabetes. Staphylococcus aureus is the most common single isolate in diabetic wounds. Human beta-defensin (hBD)-3 is antimicrobial active and appears to play a key role in the immune response. The present study aimed to analyse the effect of hBD-3 expression in a model of infected diabetic wounds. Excisional wounds were created on the backs of Yorkshire pigs and Ad5-CMV-hBD-3 vectors were microseeded. Wounds were inoculated with S. aureus, covered with a polyurethane chamber and analysed for transgene expression, bacterial infection, re-epithelialization, wound contraction, wound fluid production and blood vessel formation. hBD-3-treated wounds showed a total bacterial load of 2.1 x 10(8) colony-forming units (CFU)/g tissue, versus 1.3 x 10(9) CFU/g tissue for controls (p < 0.001) at day 4. At day 12, no statistical difference could be detected. Re-epithelialization showed 75 +/- 15% wound closure for hBD-3 expressing wounds and 50 +/- 16% for controls (p < 0.01). hBD-3 expression was in the range 15-20 ng/ml of wound fluid during day 1-4. The lower dose of 2 x 10(9) Ad5-CMV-hBD-3 showed no effect, suggesting a dose dependency for hBD-3. In the present study, we show that hBD-3 expression significantly promotes wound closure in S. aureus infected diabetic wounds in a preclinical large-animal model. Furthermore, a ten-fold reduction of bacterial growth on day 4 was detected. These findings indicate that beta-defensin-3 may play a major role in diabetic wound healing and wound infections.

274 POTENTIAL OF BOVINE MARROW STROMAL CELLS CULTURED AT DIFFERENT PASSAGES TO DIFFERENTIATE INTO ADIPOCYTE-LIKE CELLS

Bovine marrow stromal cells (MSC) would serve as a preclinical large-animal model for investigating the use of the adult pluripotent cells MSC for human cell therapies, but the information is limited. In this study, adipocytic differentiation was examined in bovine MSC at different passages (psg) after various adipogenic treatments, as the first step in characterizing the multilineage differentiation potential during an extensive culture. Bovine MSC were cultured from the femurs of 3-month-old Holstein calves using a previously described method with slight modifications (Xiang et al. 2001 Chin. J. Pathophysiol. 17, 598–601). The MSC at psg 2, 5, 10, 15, and 20 (total cell culture periods of 10, 19, 34, 49, and 64 days, respectively, 4 × 103 cells cm–2) were cultured for 2 days post-psg. Previously, we found that most of mouse MSC became adipocyte-like cells after they were cultured with a base medium (DMEM, 4.5 g L–1 glucose, plus 10% FBS) containing horse serum (HS) for 5 weeks. Therefore, the bovine MSC at each psg (70% confluence) were then cultured in the base medium containing 0.1 μm dexamethasone, 0.1 mm 3-isobutyl-1-methylxanthine, 0.2 mm indomethacin, and 10 μg mL–1 insulin [hormonal-mixture (HM) group], the base medium containing 15% HS (HS group), or the base medium (control group) for adipogenic induction. The cells were stained with oil red O after being cultured in the respective medium for 1, 3, or 5 weeks. In this study, all cells containing oil red O-stained lipid droplets (LD) were counted as ‘oil red O-positive (OP) cells’, regardless of the size and number contained. The rate of OP cells in each group was determined as follows. In the dishes, the total number of cells (60 to 100 cells) and OP cell number were counted in a randomly selected microscopic field (200×), and the rate of OP cells was calculated. A total of 10 random fields of view were examined, and the mean value was obtained for each group. Data were analyzed by ANOVA. In the HM group, OP cells were detectable about Day 7 and were clearly visible by 2 weeks. After 5 weeks, OP cell rates were significantly greater (P &lt; 0.05) in the cells at psg 2 and 5 than in those at psg 10, 15, and 20 (71 and 70% v. 66, 63, and 64%). In the HS group, cytoplasmic LD was detectable about Day 10 and the number increased gradually by 3 weeks. After 5 weeks, OP cell rate was significantly greater (P &lt; 0.05) in the cell at psg 2, 5, and 10 than in those at psg 15 and 20 (53, 57, and 51% v. 47 and 39%). Also, the size and number of LD in each cell tended to be lower in the HS group than in the corresponding HM group. In the control group, 4–5% of the cells were OP after 5 weeks. These results suggest that the ability of bovine MSC to differentiate into adipocyte-like cells decreased after long-term culture, and that horse serum was capable of inducing adipocytic differentiation of bovine MSC in vitro, but its efficacy was less than that of the hormonal mixture. Further studies to detect adipocyte-specific markers, such as G3PDH activity and aP2 expression, from these cells are needed for the quantitative analysis of the differentiation.

The Use of an Approved Biodegradable Polymer Scaffold as a Solid Support System for Improvement of Islet Engraftment

The aim of this study was to investigate whether the use of a medically approved biodegradable scaffold as a solid support system would enhance graft survival following transplantation into the omental pouch in a preclinical large animal model. Six beagle dogs underwent total pancreatectomy followed by islet autotransplantation into the omental pouch. Four dogs received islets seeded in a biodegradable polymer scaffold and two received free islets without a scaffold. All four animals that received islets in the scaffold became normoglycemic without exogeneous insulin injection. One dog, transplanted with the largest number of islets, maintained a normal metabolic state until the graft was removed at 5 months posttransplant. In two out of the three that received a marginal islet mass, insulin independence was sustained up to 2 months. In contrast, two dogs transplanted with a similar marginal mass without the scaffold never became normoglycemic. Histological examination of the grafts in the scaffold showed numerous well-granulated, insulin-containing cells as well as glucagon-positive cells. These results indicate that biodegradable scaffolds may enhance survival and function of islet grafts. Manipulation of the microenvironment of transplanted islets may constitute the basis for new approaches to enhance islet engraftment.

Insulin‐like growth factor‐1 gene therapy and cell transplantation in diabetic wounds

Impaired wound healing is a frequent phenomenon in diabetes mellitus. However, little is known of the fundamental cause of this pathology. The present study examined the effect of human insulin-like growth factor (hIGF)-1 overexpression in combination with autologous cell transplantation to diabetic wounds in a preclinical large-animal model. Diabetes was induced in Yorkshire pigs with streptozotocin. Keratinocytes were cultured and transfected with hIGF-1 or LacZ transgene. Plasmids were lipoplexed with either Lipofectin or Lipofectamin 2000. Transgene expression was assessed by enzyme-linked immunosorbent assay or X-gal staining. For in vivo studies, full-thickness wounds were created and dressed with a sealed chamber. Transfected cells were transplanted into the wounds. Wound contraction was monitored and biopsies were obtained for measurement of re-epithelialization. Wound fluid was collected and analysed for IGF-1 concentrations. Quantification showed up to 740 ng/ml IGF-1 in vitro and significantly higher concentrations over 14 days compared to controls for the Lipofectamin 2000 group. Lipofectin-mediated gene transfer showed peak expression on day 2 with 68.5 ng/ml. In vivo, transfected cells showed peak expression of 457 ng/ml at day 1, followed by subsequent decline to 5 ng/ml on day 12 with Lipofectamin 2000. For Lipofectin, no significant IGF-1 expression could be detected. Gene therapy caused significantly faster wound closure (83%) than both controls (native-cell therapy = 57%; control wounds = 32%). The present study demonstrates that optimized nonviral gene transfer increased IGF-1 expression in diabetic wounds by up to 900-fold. This high IGF-1 concentration in combination with cell therapy improved diabetic wound healing significantly.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays

Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with 3H for MLR and 51Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. Objective The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.

Reversal of Cardiac Dysfunction After Long-Term Expression of SERCA2a by Gene Transfer in a Pre-Clinical Model of Heart Failure

The aim of this study was to examine the effects of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model. Reduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known. Yorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2). At 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 +/- 3.2 s(-1) group 1 vs. 15.5 +/- 3.0 s(-1) group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs -3.0 +/- 10% vs. +15 +/- 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis. Using a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling.

Stem cells in the heart: What's the buzz all about? Part 2: Arrhythmic risks and clinical studies

New approaches for cardiac repair have been enabled by the discovery that the heart contains its own reservoir of stem cells. In Part 1 of this review, we discussed various cardiac stem cell populations, reviewed our own work on cardiosphere-derived cells from human hearts, and outlined large animal preclinical models testing the regenerative potential of cardiac stem cells. Here we continue with a discussion on other adult stem cell sources with clinical potential. We summarize the critical safety issues associated with stem cell therapy and present the possible proarrhythmic and antiarrhythmic effects of stem cell transplantation. We discuss the outcomes of clinical stem cell trials and identify the technical, ethical, and practical issues facing the clinical application of cardiac stem cells.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells.

Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.