Laparoscopy for the Treatment of Congenital Hernia: Use of Surgical Meshes and Mesenchymal Stem Cells in a Clinically Relevant Animal Model.

More than a century has passed since the first surgical mesh for hernia repair was developed, and, to date, this is still the most widely used method despite the great number of complications it poses. The purpose of this study was to combine stem cell therapy and laparoscopy for the treatment of congenital hernia in a swine animal model. Porcine bone marrow-derived mesenchymal stem cells (MSCs) were seeded on polypropylene surgical meshes using a fibrin sealant solution as a vehicle. Meshes with (cell group) or without (control group) MSCs were implanted through laparoscopy in Large White pigs with congenital abdominal hernia after the approximation of hernia borders (implantation day). A successive laparoscopic biopsy of the mesh and its surrounding tissues was performed a week after implantation, and surgical meshes were excised a month after implantation. Ultrasonography was used to measure hernia sizes. Flow cytometry, histological, and gene expression analyses of the biopsy and necropsy samples were performed. The fibrin sealant solution was easy to prepare and preserved the viability of MSCs in the surgical meshes. Ultrasonography demonstrated a significant reduction in hernia size 1 week after implantation in the cell group relative to that on the day of implantation (p < 0.05). Flow cytometry of the mesh-infiltrated cells showed a non-significant increase of M2 macrophages when the cell group was compared with the control group 1 week after implantation. A significant decrease in the gene expression of VEGF and a significant increase in TNF expression were determined in the cell group 1 month after implantation compared with gene expressions in the control group (p < 0.05). Here, we propose an easy and feasible method to combine stem cell therapy and minimally invasive surgical techniques for hernia repair. In this study, stem cell therapy did not show a great immunomodulatory or regenerative effect in overcoming hernia-related complications. However, our clinically relevant animal model with congenital hernia closely resembles the clinical human condition. Further studies should be focused on this valuable animal model to evaluate stem cell therapies in hernia surgery.

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Dose-dependent improvement of cardiac function in a swine model of acute myocardial infarction after intracoronary administration of allogeneic heart-derived cells.

BACKGROUND: Allogeneic cardiac-derived progenitor cells (CPC) without immunosuppression could provide an effective ancillary therapy to improve cardiac function in reperfused myocardial infarction. We set out to perform a comprehensive preclinical feasibility and safety evaluation of porcine CPC (pCPC) in the infarcted porcine model, analyzing biodistribution and mid-term efficacy, as well as safety in healthy non-infarcted swine. METHODS: The expression profile of several pCPC isolates was compared with humans using both FACS and RT-qPCR. ELISA was used to compare the functional secretome. One week after infarction, female swine received an intracoronary (IC) infusion of vehicle (CON), 25 × 106 pCPC (25 M), or 50 × 106 pCPC (50 M). Animals were followed up for 10 weeks using serial cardiac magnetic resonance imaging to assess functional and structural remodeling (left ventricular ejection fraction (LVEF), systolic and diastolic volumes, and myocardial salvage index). Statistical comparisons were performed using Kruskal-Wallis and Mann-Whitney U tests. Biodistribution analysis of 18F-FDG-labeled pCPC was also performed 4 h after infarction in a different subset of animals. RESULTS: Phenotypic and functional characterization of pCPC revealed a gene expression profile comparable to their human counterparts as well as preliminary functional equivalence. Left ventricular functional and structural remodeling showed significantly increased LVEF 10 weeks after IC administration of 50 M pCPC, associated to the recovery of left ventricular volumes that returned to pre-infarction values (LVEF at 10 weeks was 42.1 ± 10.0% in CON, 46.5 ± 7.4% in 25 M, and 50.2 ± 4.9% in 50 M, p < 0.05). Infarct remodeling was also improved following pCPC infusion with a significantly higher myocardial salvage index in both treated groups (0.35 ± 0.20 in CON; 0.61 ± 0.20, p = 0.04, in 25 M; and 0.63 ± 0.17, p = 0.01, in 50 M). Biodistribution studies demonstrated cardiac tropism 4 h after IC administration, with substantial myocardial retention of pCPC-associated tracer activity (18% of labeled cells in the heart), and no obstruction of coronary flow, indicating their suitability as a cell therapy product. CONCLUSIONS: IC administration of allogeneic pCPC at 1 week after acute myocardial infarction is feasible, safe, and associated with marked structural and functional benefit. The robust cardiac tropism of pCPC and the paracrine effects on left ventricle post-infarction remodeling established the preclinical bases for the CAREMI clinical trial (NCT02439398).

Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8-12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

Ischemia-reperfusion injury in a rat microvascular skin free flap model: A histological, genetic, and blood flow study.

Ischemia reperfusion injury is associated with tissue damage and inflammation, and is one of the main factors causing flap failure in reconstructive microsurgery. Although ischemia-reperfusion (I/R) injury is a well-studied aspect of flap survival, its biological mechanisms remain to be elucidated. To better understand the biological processes of ischemia reperfusion injury, and to develop further therapeutic strategies, the main objective of this study was to identify the gene expression pattern and histological changes in an I/R injury animal model. Fourteen rats (n = 7/group) were randomly divided into control or ischemia-reperfusion group (8 hours of ischemia). Microsurgical anastomoses were objectively assessed using transit-time-ultrasound technology. Seven days after surgery, flap survival was evaluated and tissue samples were harvested for anatomopathological and gene-expression analyses.The I/R injury reduced the survival of free flaps and histological analyses revealed a subcutaneous edema together with an inflammatory infiltrate. Interestingly, the Arginase 1 expression level as well as the ratio of Arginase 1/Nitric oxide synthase 2 showed a significant increase in the I/R group. In summary, here we describe a well-characterized I/R animal model that may serve to evaluate therapeutic agents under reproducible and controlled conditions. Moreover, this model could be especially useful for the evaluation of arginase inhibitors and different compounds of potential interest in reconstructive microsurgery.

Extracellular vesicles derived from endometrial human mesenchymal stem cells improve IVF outcome in an aged murine model.

Advanced age reduces the success of in vitro fertilization (IVF) being this effect partly mediated by an overproduction of reactive oxygen species (ROS) that trigger apoptosis. It has been demonstrated that extracellular vesicles derived from endometrial mesenchymal stem cells (EV-endMSCs) exert an antioxidant effect and can be used as IVF coadjutants. In this work, endMSCs were isolated from human menstrual blood (n = 4) and characterized according to multipotentiality and surface marker expression prior EV-endMSCs isolation. Oocytes were obtained from 21 B6D2 mice (24 weeks) and coincubated with sperm from young males (8-12 weeks). Presumptive zygotes were incubated in the presence of 0, 10, 20, 40 or 80 µg/ml of EV-endMSCs in KSOM medium. Blastocyst yield was evaluated, and 25 blastocysts per group were used for qPCR. Blastocyst rate was 29.4% in control; 45.2% for 10 µg/ml, 62.9% for 20 µg/ml, 55.5% for 40 µg/ml and 53.8% in the 80 µg/ml (n = 124-130 oocytes) being all the increases significantly different when compared against control (p < 0.05). The 20-80 µg/ml treatments decreased the expression of glutathione peroxidase (Gpx1), and the 10-40 µg/ml treatments reduced the expression of superoxide dismutase (Sod1; p < 0.05) compared to control; Bax mRNA expression did not vary. Our results suggest that the increased developmental competence of the embryos could be partly mediated by the EV-endMSCs' ROS scavenger activity.

The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta.

Endometrial mesenchymal stem cells (endMSCs) reside in the basal and functional layer of human endometrium and participate in tissue remodelling, which is required for maintaining the regenerative capacity of the endometrium. The endMSCs are multipotent stem cells and exhibit immunomodulatory effects. This paper aimed to evaluate the regulatory effects of extracellular vesicles derived from endMSCs (EV-endMSCs) in the setting of T cell activation. In vitro stimulations of lymphocytes were performed in the presence of EV-endMSCs. These in vitro-stimulated lymphocytes were functionally and phenotypically characterized to distinguish CD4+ and CD8+ T cell differentiation subsets. Moreover, the inhibition of TGFß was performed with neutralizing antibodies. The phenotype and nanoparticle tracking analysis of the EV-endMSCs demonstrated that they are similar in terms of size distribution to other mesenchymal stem cells-derived exosomes. The in vitro assays showed an immunomodulatory potential of these vesicles to counteract the differentiation of CD4+ T cells. The quantification of active TGFß in EV-endMSCs was found to be very high when compared with extracellular vesicles-free concentrated supernatants. Finally, the neutralization of TGFß significantly attenuated the immunomodulatory activity of EV-endMSCs. In summary, this is the first report demonstrating that EV-endMSCs exhibit a potent inhibitory effect against CD4+ T cell activation, which is partially mediated by TGFß signalling.

Adipose-Derived Stem Cells Ameliorate Ischemia-Reperfusion Injury in a Rat Skin Free Flap Model.

BACKGROUND: Ischemia-reperfusion (I/R) injury is inevitable during free tissue transfers. When the period of ischemia exceeds the tissue tolerance, it causes necrosis and flap failure. The aim of this study was to investigate the effects of adipose-derived stem cells (ASCs) embedded in a collagen type I scaffold on the survival of free skin flaps to counteract I/R injury. METHODS: Left superficial caudal epigastric skin flaps (3 × 6 cm) were performed in 28 Wistar rats that were divided into four groups. The flaps elevated in the animals of the control group did not suffer any ischemic insult, and the vascular pedicle was not cut. All other flaps were subjected to 8 hours of ischemia prior to revascularization: I/R control group (8 hours of ischemia), I/R scaffold group (8 hours of ischemia + collagen type I scaffold), and I/R scaffold-ASCs group (8 hours of ischemia + collagen type I scaffold with rat ASCs embedded). Transit-time ultrasound blood flow measurements were performed. After 7 days, the areas of flap survival were measured and tissues were stained with hematoxylin/eosin and Masson's trichrome stain for histological analysis. RESULTS: The mean percentage flap survival area was significantly higher in the ASCs-treated flaps (I/R scaffold-ASCs group) compared with the ischemic controls (I/R control group and I/R scaffold group). Higher vascular proliferation and lower severity of necrosis and inflammatory changes were seen histologically in the samples of the ASCs-treated group. No significant difference in blood flow was detected between groups. CONCLUSION: Subcutaneous administration of ASCs embedded on a collagen type I scaffold reduces tissue damage after I/R injury in microvascular free flaps.

Murine embryos exposed to human endometrial MSCs-derived extracellular vesicles exhibit higher VEGF/PDGF AA release, increased blastomere count and hatching rates.

Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst's total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development.

Fibrin glue mesh fixation combined with mesenchymal stem cells or exosomes modulates the inflammatory reaction in a murine model of incisional hernia.

Surgical meshes are effective and frequently used to reinforce soft tissues. Fibrin glue (FG) has been widely used for mesh fixation and is also considered an optimal vehicle for stem cell delivery. The aim of this preclinical study was to evaluate the therapeutic effect of MSCs and their exosomes combined with FG for the treatment of incisional hernia. A murine incisional hernia model was used to implant surgical meshes and different treatments with FG, MSCs and exo-MSCs were applied. The implanted meshes were evaluated at day 7 by anatomopathology, cellular analysis of infiltrating leukocytes and gene expression analysis of TH1/TH2 cytokines, MMPs, TIMPs and collagens. Our results demonstrated a significant increase of anti-inflammatory M2 macrophages and TH2 cytokines when MSCs or exo-MSCs were used. Moreover, the analysis of MMPs, TIMPs and collagen exerted significant differences in the extracellular matrix and in the remodeling process. Our in vivo study suggests that the fixation of surgical meshes with FG and MSCs or exo-MSCs will have a beneficial effect for the treatment of incisional hernia in terms of improved outcomes of damaged tissue, and especially, in the modulation of inflammatory responses towards a less aggressive and pro-regenerative profile. STATEMENT OF SIGNIFICANCE: The implantation of surgical meshes is the standard procedure to reinforce tissue defects such as hernias. However, an exacerbated and persistent inflammatory response secondary to this implantation is frequently observed, leading to a strong discomfort and chronic pain in the patients. In many cases, an additional surgical intervention is needed to remove the mesh. This study shows that mesenchymal stem cells and their exosomes, combined with a fibrin sealant, can be used for the successful fixation of these meshes. This new therapeutic approach, assayed in a murine model of incisional hernia, favors the modulation of the inflammatory response towards a less aggressive and pro-regenerative profile.

Evaluation of antigen-induced synovitis in a porcine model: Immunological, arthroscopic and kinetic studies.

BACKGROUND: Synovitis is an inflammation-related disease linked to rheumatoid arthritis, osteoarthritis, infections and trauma. This inflammation is accompanied by immune cells infiltration which initiates an inflammatory response causing pain, discomfort and affecting the normal joint function. The treatment of synovitis is based on the administration of anti-inflammatory drugs or biological agents such as platelet rich plasma and mesenchymal stem cells. However, the evaluation and validation of more effective therapies of synovitis requires the establishment of clinically relevant animal models. RESULTS: In this study, Large White pigs were pre-immunized to evaluate an antigen-induced synovitis. The immune monitoring of synovial fluids in this model allowed us the identification of IL-12p40 and T cell subsets as immune biomarkers. Moreover, the evolution of synovitis was performed by arthroscopic procedures and kinetic analysis. In summary, this paper describes an animal model of antigen-induced synovitis to be used in the evaluation of anti-inflammatory therapies. CONCLUSIONS: The novelty of this paper lies in the development of a clinically relevant model of synovitis which permits the simultaneous evaluation of synovitis from an immunological, surgical and kinetic point of view.

Comparison of mesenchymal stem cells and leukocytes from Large White and Göttingen Minipigs: Clues for stem cell-based immunomodulatory therapies.

The mesenchymal stem cells (MSCs) are one of the most promising cell types for human and veterinary use and their therapeutic effect is associated with their immunomodulatory properties. Farm animal models, such as pigs, have become a valuable tool to evaluate the safety and efficacy of adoptively transferred MSCs in the setting of veterinary medicine. In order to evaluate the immunomodulatory effect of stem cell-based therapies in porcine breeds, a deep analysis and comparison of MSCs and leukocyte subsets are absolutely necessary. Here we provide a detailed analysis of bone-marrow derived MSCs and leukocyte subsets from Large White pigs and Göttingen Minipigs. Significant differences were observed between the two pig breeds in terms of T cell subsets that need to be considered for immune monitoring of stem cell-based therapies.

Intrapericardial Delivery of Cardiosphere-Derived Cells: An Immunological Study in a Clinically Relevant Large Animal Model.

INTRODUCTION: The intrapericardial delivery has been defined as an efficient method for pharmacological agent delivery. Here we hypothesize that intrapericardial administration of cardiosphere-derived cells (CDCs) may have an immunomodulatory effect providing an optimal microenvironment for promoting cardiac repair. To our knowledge, this is the first report studying the effects of CDCs for myocardial repair using the intrapericardial delivery route. MATERIAL AND METHODS: CDCs lines were isolated, expanded and characterized by flow cytometry and PCR. Their differentiation ability was determined using specific culture media and differential staining. 300,000 CDCs/kg were injected into the pericardial space of a swine myocardial infarcted model. Magnetic resonance imaging, biochemical analysis of pericardial fluid and plasma, cytokine measurements and flow cytometry analysis were performed. RESULTS: Our results showed that, phenotype and differentiation behavior of porcine CDCs were equivalent to previously described CDCs. Moreover, the intrapericardial administration of CDCs fulfilled the safety aspects as non-adverse effects were reported. Finally, the phenotypes of resident lymphocytes and TH1 cytokines in the pericardial fluid were significantly altered after CDCs administration. CONCLUSIONS: The pericardial fluid could be considered as a safe and optimal vehicle for CDCs administration. The observed changes in the studied immunological parameters could exert a modulation in the inflammatory environment of infarcted hearts, indirectly benefiting the endogenous cardiac repair.

Surgical meshes coated with mesenchymal stem cells provide an anti-inflammatory environment by a M2 macrophage polarization.

Surgical meshes are widely used in clinics to reinforce soft tissue's defects, and to give support to prolapsed organs. However, the implantation of surgical meshes is commonly related with an inflammatory response being difficult to eradicate without removing the mesh. Here we hypothesize that the combined use of surgical meshes and mesenchymal stem cells (MSCs) could be a useful tool to reduce the inflammatory reaction secondary to mesh implantation. In vitro determinations of viability, metabolic activity and immunomodulation assays were performed on MSCs-coated meshes. Magnetic resonance imaging, evaluation by laparoscopic optical system and histology were performed for safety assessment. Finally, flow cytometry and qRT-PCR were used to elucidate the mechanism of action of MSCs-coated meshes. Our results demonstrate the feasibility to obtain MSCs-coated surgical meshes and their cryopreservability to be used as an 'off the shelf' product. These biological meshes fulfill the safety aspects as non-adverse effects were observed when compared to controls. Moreover, both in vitro and in vivo studies demonstrated that, local immunomodulation of implanted meshes is mediated by a macrophage polarization towards an anti-inflammatory phenotype. In conclusion, the combined usage of surgical meshes with MSCs fulfills the safety requirements for a future clinical application, providing an anti-inflammatory environment that could reduce the inflammatory processes commonly observed after surgical mesh implantation. STATEMENT OF SIGNIFICANCE: Surgical meshes are medical devices widely used in clinics to resolve hernias and organs' prolapses, among other disorders. However, the implantation of surgical meshes is commonly related with an inflammatory response being difficult to eradicate without removing the mesh, causing pain and discomfort in the patient. Previously, the anti-inflammatory, immunomodulatory and pro-regenerative ability of mesenchymal stem cells (MSCs) have been described. To our knowledge, this is the first report where the anti-inflammatory and pro-regenerative ability of MSCs have been successfully applied in combination with surgical meshes, reducing the inflammatory processes commonly observed after mesh implantation. Moreover, our in vitro and in vivo results highlight the safety and efficacy of these bioactive meshes as a 'ready to use' medical product.

Assessment of the Behavior of Mesenchymal Stem Cells Immobilized in Biomimetic Alginate Microcapsules.

The combination of mesenchymal stem cells (MSCs) and biomimetic matrices for cell-based therapies has led to enormous advances, including the field of cell microencapsulation technology. In the present work, we have evaluated the potential of genetically modified MSCs from mice bone marrow, D1-MSCs, immobilized in alginate microcapsules with different RGD (Arg-Gly-Asp) densities. Results demonstrated that the microcapsules represent a suitable platform for D1-MSC encapsulation since cell immobilization into alginate matrices does not affect their main characteristics. The in vitro study showed a higher activity of D1-MSCs when they are immobilized in RGD-modified alginate microcapsules, obtaining the highest therapeutic factor secretion with low and intermediate densities of the bioactive molecule. In addition, the inclusion of RGD increased the differentiation potential of immobilized cells upon specific induction. However, subcutaneous implantation did not induce differentiation of D1-MSCs toward any lineage remaining at an undifferentiated state in vivo.

Estudio comparativo del aislamiento de exosomas derivados de células madre mesenquimales humanas para uso clínico / Comparative study of isolated human mesenchymal stem cell derived exosomes for clinical use / Estudo comparativo do isolamento de exossomos derivados de celulas-tronco mesenquimais humanas para uso clinico

Los exosomas son microvesículas derivadas de la exocitosis que son liberados al espacio extracelular. Las funciones de los exosomas dependen en gran medida de la célula de la cual provienen. Los exosomas derivados de células madre mesenquimales (Mesenchymal Stem Cells, MSCs), al igual que las células de origen, poseen un enorme potencial terapéutico que favorece la regeneración tisular y reduce la inflamación. Los prometedores resultados preclínicos que emplean estos exosomas han abierto las puertas a la futura aplicación clínica de estas vesículas. El diseño de nuevos protocolos que permitan el aislamiento de exosomas para su aplicación clínica es una necesidad actual teniendo en cuenta el enorme interés que ha surgido a raíz de los prometedores resultados en ensayos preclínicos. El objetivo de este trabajo ha sido comparar, en términos de rendimiento, tamaño y pureza, diferentes métodos de aislamiento de exosomas a partir de MSCs humanas. Los resultados obtenidos demuestran que el uso de filtros concentradores para sobrenadantes de cultivos podría ser una alternativa a los protocolos convencionales basados en la ultracentrifugación. Los resultados de este trabajo permiten orientar en el diseño de protocolos para la obtención de exosomas derivados de MSCs en grado clínico...

Delayed administration of allogeneic cardiac stem cell therapy for acute myocardial infarction could ameliorate adverse remodeling: experimental study in swine.

BACKGROUND: The optimal timing of cardiac stem cells administration is still unclear. We assessed the safety of same-day and delayed (one week) delivery and the possible influence of the timing on the therapeutic outcomes of allogeneic porcine cardiac stem cells administration after acute myocardial infarction in a closed-chest ischemia-reperfusion model. METHODS: Female swine surviving 90 min occlusion of the mid left anterior descending coronary artery received an intracoronary injection of 25x10(6) porcine cardiac stem cells either two hours (n = 5, D0) or 7 days (n = 6, D7) after reperfusion. Controls received intracoronary injection of vehicle on day 7 (n = 6, CON). Safety was defined in terms of absence of major cardiac events, changes to the ECG during injection, post-administration coronary flow assessed using the TIMI scale and cardiac troponin I determination after the intervention. Cardiac Magnetic Resonance was performed for morphological and functional assessment prior to infarction, before injection (D7 and CON groups only), at one and 10 weeks. Samples were taken from the infarct and transition areas for pathological examination. RESULTS: No major adverse cardiac events were seen during injection in any group. Animals receiving the therapy on the same day of infarction (D0 group) showed mild transient ST changes during injection (n = 4) and, in one case, slightly compromised coronary flow (TIMI 2). Cardiac function parameters and infarct sizes were not significantly different between groups, with a trend towards higher ejection fraction in the treated groups. Ventricular volumes indexed to body surface area increased over time in control animals, and decreased by the end of the study in animals receiving the therapy, significantly so when comparing End Diastolic Volume between CON and D7 groups (CON: 121.70 ml/m(2) ± 26.09 ml/m(2), D7: 98.71 ml/m(2) ± 8.30 ml/m(2), p = 0.037). The treated groups showed less organization of the collagenous scar, and a significantly (p = 0.019) higher amount of larger, more mature vessels at the infarct border. CONCLUSIONS: The intracoronary injection of 25x10(6) allogeneic cardiac stem cells is generally safe, both early and 7 days after experimental infarction, and alleviates myocardial dysfunction, with a greater limitation of left ventricular remodeling when performed at one week.

Intrapericardial administration of mesenchymal stem cells in a large animal model: a bio-distribution analysis.

The appropriate administration route for cardiovascular cell therapy is essential to ensure the viability, proliferative potential, homing capacity and implantation of transferred cells. At the present, the intrapericardial administration of pharmacological agents is considered an efficient method for the treatment of cardiovascular diseases. However, only a few reports have addressed the question whether the intrapericardial delivery of Mesenchymal Stem Cells (MSCs) could be an optimal administration route. This work firstly aimed to analyze the pericardial fluid as a cell-delivery vehicle. Moreover, the in vivo biodistribution pattern of intrapericardially administered MSCs was evaluated in a clinically relevant large animal model. Our in vitro results firstly showed that, MSCs viability, proliferative behavior and phenotypic profile were unaffected by exposure to pericardial fluid. Secondly, in vivo cell tracking by magnetic resonance imaging, histological examination and Y-chromosome amplification clearly demonstrated the presence of MSCs in pericardium, ventricles (left and right) and atrium (left and right) when MSCs were administered into the pericardial space. In conclusion, here we demonstrate that pericardial fluid is a suitable vehicle for MSCs and intrapericardial route provides an optimal retention and implantation of MSCs.

Allogeneic cardiac stem cell administration for acute myocardial infarction.

Myocardial infarction, even after reperfusion, leads to significant loss of cardiomyocytes and to a maladaptive remodeling process. A possibility gaining attention as an ancillary therapy is the use of cardiac-derived cell products, with early stage clinical trials reporting highly promising results with autologous cells. However, an autologous therapy presents limitations, such as timeframe of therapy, cell processing and culture costs, risks posed to the patient by the tissue harvesting, etc. Allogeneic cells may represent an answer, providing an off-the-shelf product that could be used in the acute stage, before the myocardial damage is irrevocable. To date, allogeneic cardiac-derived cell products are being tested extensively, but the questions of their immunogenicity (and therefore safety), efficacy, cost-effectiveness, etc. are only partially elucidated. Small Phase I/II clinical trials (ALLSTAR, CAREMI) have started and their results will shed the much needed light on the feasibility and safety of a much needed therapy.

Mesenchymal stem cell-coated sutures enhance collagen depositions in sutured tissues.

Sutures are commonly used for surgical procedures and new sutures are being developed to improve wound healing. In the past decade, it has been extensively shown that mesenchymal stem cells (MSCs) have a wound healing potential. To benefit the overall wound healing process, we aimed to analyze the usage of pretreated sutures for improving the implantation of MSCs in the tissues. Our results firstly showed that suture pretreatments with gelatin, poly-L-lysine, and NaOH improved the adhesive strength of MSCs to sutures. These cells remained surrounding the sutured tissue and no significant phenotypic changes were found in those cells cultured onto pretreated sutures. In vivo experiments showed that the implantation of MSCs by suturing increases the collagen content in the sutured tissue. Moreover, proteomics analysis of secreted proteins showed that collagen alpha-1(I) chain was the most abundant collagen found. To our knowledge, this is the first report that aimed to improve the implantation of MSCs in tissue by suture pretreatments. Moreover, in vivo experiments suggest that MSC-coated sutures may enhance wound healing and tissue remodeling through the release of different collagen types being applicable for those patients that tend to have difficulty healing.