Enhanced expression of natural cytotoxicity receptors on cytokine-induced memory-like natural killer cells correlates with effector function.

Introduction: Natural killer (NK) cells are a key component of the innate immune system, involved in defending the host against virus-infected cells and tumor immunosurveillance. Under in vitro culture conditions, IL-12/15/18 can induce a memory-like phenotype in NK cells. These cytokine-induced memory-like (CIML) NK cells possess desirable characteristics for immunotherapies, including a longer lifespan and increased cytotoxicity. Methods: In this study, NK cells were isolated from peripheral blood of healthy donors and stimulated with IL-12/15/18 to induce a memory-like phenotype or with IL-15 alone as a control. After seven days of culture, multiparametric flow cytometry analysis was performed to evaluate the phenotypic and functional profiles of CIML and control NK cells. Results: Our results showed a significantly higher expression of CD25, CD69, NKG2D, NKp30, NKp44, NKp46, TACTILE, and Granzyme B in CIML NK cells compared to control NK cells. In contrast, KIR2D expression was significantly lower in CIML NK cells than in control NK cells. Moreover, functional experiments demonstrated that CIML NK cells displayed enhanced degranulation capacity and increased intracellular IFN-γ production against the target cell line K562. Interestingly, the degranulation capacity of CIML NK cells was positively correlated with the expression of the activating receptors NKp46 and NKp30, as well as with the inhibitory receptor TACTILE. Discussion: In conclusion, this study provides a deep phenotypic characterization of in vitro-expanded CIML NK cells. Moreover, the correlations found between NK cell receptors and degranulation capacity of CIML NK cells allowed the identification of several biomarkers that could be useful in clinical settings.

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

Menstrual blood-derived stromal cells: insights into their secretome in acute hypoxia conditions.

BACKGROUND: Despite constant advances in regenerative medicine, the closure of chronic wounds is still challenging. Therapeutic approaches using locally administered MSCs have been considered a promising option. However, the viability of these cells is seriously threatened by acute hypoxic stress linked to wound healing. In this work, we aimed to study the tolerance of Menstrual blood-derived stromal cells (MenSCs) to acute hypoxia and their therapeutic paracrine effect. METHODS: Isolated MenSCs were phenotypically characterized and evaluated in terms of proliferation, viability, and gene expression, under acute hypoxia (AH) compared with conventional cultured condition or normoxia (N). A step further, the secretome of MenSCs under acute hypoxia was analyzed with respect to their miRNAs content and by in vitro functional assays. For the analysis of differences between the two groups, Student's t-test was performed and one-way ANOVA and Tukey's multiple comparisons test for multiple groups were used. RESULTS: Our results revealed that the viability of MenSCs was not affected under acute hypoxia, although proliferation rate slowed down. Gene analysis revealed 5 up-regulated (BNIP3, ANGPTL4, IL6, IL1B, and PDK1) and 4 down-regulated genes (IDO1, HMOX1, ANGPTL2, and HGF) in AH compared to N. Global gene expression analysis revealed a decrease in the gene ontology functions of migration and wound response with respect to the normoxic condition. In contrast, functions such as angiogenesis were enriched under the AH condition. Regarding the secretome analysis, two miRNAs involved in angiogenic processes (hsa-miR-148a-3p and hsa-miR-378a-3p), were significantly up-expressed when compared to the normoxic condition, being MYC gene, the unique target of both. Functional assays on HUVECs revealed a potential pro-angiogenic capacity of MenSCs cultured in both oxygen conditions (N and AH) based on the wound closure and tube formation results of their released paracrine factors. However, when compared to normoxia, the paracrine factors of MenSCs under acute hypoxia slightly reduced the proliferation, migration, and in vitro wound closure of HUVECs. CONCLUSIONS: MenSC exhibited a good survival capacity under acute hypoxic conditions as well as beneficial properties applicable in the field of tissue regeneration through their secretome, which makes them a potential cell source for wound healing interventions.

Transcriptome Profile Reveals Differences between Remote and Ischemic Myocardium after Acute Myocardial Infarction in a Swine Model.

Acute myocardial infarction (AMI) is the consequence of an acute interruption of myocardial blood flow delimiting an area with ischemic necrosis. The loss of cardiomyocytes initiates cardiac remodeling in the myocardium, leading to molecular changes in an attempt to recover myocardial function. The purpose of this study was to unravel the differences in the molecular profile between ischemic and remote myocardium after AMI in an experimental model. To mimic human myocardial infarction, healthy pigs were subjected to occlusion of the mid-left anterior descending coronary artery, and myocardial tissue was collected from ischemic and remote zones for omics techniques. Comparative transcriptome analysis of both areas was accurately validated by proteomic analysis, resulting in mitochondrion-related biological processes being the most impaired mechanisms in the infarcted area. Moreover, Immune system process-related genes were up-regulated in the remote tissue, mainly due to the increase of neutrophil migration in this area. These results provide valuable information regarding differentially expressed genes and their biological functions between ischemic and remote myocardium after AMI, which could be useful for establishing therapeutic targets for the development of new treatments.

Intracoronary Administration of Microencapsulated HGF in a Reperfused Myocardial Infarction Swine Model.

Therapy microencapsulation allows minimally invasive, safe, and effective administration. Hepatocyte growth factor (HGF) has angiogenic, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties. Our objective was to evaluate the cardiac safety and effectiveness of intracoronary (IC) administration of HGF-loaded extended release microspheres in an acute myocardial infarction (AMI) swine model. An IC infusion of 5 × 106 HGF-loaded microspheres (MS+HGF, n = 7), 5 × 106 placebo microspheres (MS, n = 7), or saline (SAL, n = 7) was performed two days after AMI. TIMI flow and Troponin I (TnI) values were assessed pre- and post-treatment. Cardiac function was evaluated with magnetic resonance imaging (cMR) before injection and at 10 weeks. Plasma cytokines were determined to evaluate the inflammatory profile and hearts were subjected to histopathological evaluation. Post-treatment coronary flow was impaired in five animals (MS+HGF and MS group) without significant increases in TnI. One animal (MS group) died during treatment. There were no significant differences between groups in cMR parameters at any time (p > 0.05). No statistically significant changes were found between groups neither in cytokines nor in histological analyses. The IC administration of 5 × 106 HGF-loaded-microspheres 48 h post-AMI did not improve cardiac function, nor did it decrease inflammation or cardiac fibrosis in this experimental setting.

Effects of Cardiac Stem Cell on Postinfarction Arrhythmogenic Substrate.

Clinical data suggest that cardiosphere-derived cells (CDCs) could modify post-infarction scar and ventricular remodeling and reduce the incidence of ventricular tachycardia (VT). This paper assesses the effect of CDCs on VT substrate in a pig model of postinfarction monomorphic VT. We studied the effect of CDCs on the electrophysiological properties and histological structure of dense scar and heterogeneous tissue (HT). Optical mapping and histological evaluation were performed 16 weeks after the induction of a myocardial infarction by transient occlusion of the left anterior descending (LAD) artery in 21 pigs. Four weeks after LAD occlusion, pigs were randomized to receive intracoronary plus trans-myocardial CDCs (IC+TM group, n: 10) or to a control group. Optical mapping (OM) showed an action potential duration (APD) gradient between HT and normal tissue in both groups. CDCs increased conduction velocity (53 ± 5 vs. 45 ± 6 cm/s, p < 0.01), prolonged APD (280 ± 30 ms vs. 220 ± 40 ms, p < 0.01) and decreased APD dispersion in the HT. During OM, a VT was induced in one and seven of the IC+TM and control hearts (p = 0.03), respectively; five of these VTs had their critical isthmus located in intra-scar HT found adjacent to the coronary arteries. Histological evaluation of HT revealed less fibrosis (p < 0.01), lower density of myofibroblasts (p = 0.001), and higher density of connexin-43 in the IC+TM group. Scar and left ventricular volumes did not show differences between groups. Allogeneic CDCs early after myocardial infarction can modify the structure and electrophysiology of post-infarction scar. These findings pave the way for novel therapeutic properties of CDCs.

Intrapericardial Administration of Secretomes from Menstrual Blood-Derived Mesenchymal Stromal Cells: Effects on Immune-Related Genes in a Porcine Model of Myocardial Infarction.

Acute myocardial infarction (AMI) is a manifestation of ischemic heart disease where the immune system plays an important role in the re-establishment of homeostasis. We hypothesize that the anti-inflammatory activity of secretomes from menstrual blood-derived mesenchymal stromal cells (S-MenSCs) and IFNγ/TNFα-primed MenSCs (S-MenSCs*) may be considered a therapeutic option for the treatment of AMI. To assess this hypothesis, we have evaluated the effect of S-MenSCs and S-MenSCs* on cardiac function parameters and the involvement of immune-related genes using a porcine model of AMI. Twelve pigs were randomly divided into three biogroups: AMI/Placebo, AMI/S-MenSCs, and AMI/S-MenSCs*. AMI models were generated using a closed chest coronary occlusion-reperfusion procedure and, after 72 h, the different treatments were intrapericardially administered. Cardiac function parameters were monitored by magnetic resonance imaging before and 7 days post-therapy. Transcriptomic analyses in the infarcted tissue identified 571 transcripts associated with the Gene Ontology term Immune response, of which 57 were differentially expressed when different biogroups were compared. Moreover, a prediction of the interactions between differentially expressed genes (DEGs) and miRNAs from secretomes revealed that some DEGs in the infarction area, such as STAT3, IGFR1, or BCL6 could be targeted by previously identified miRNAs in secretomes from MenSCs. In conclusion, the intrapericardial administration of secretome early after infarction has a significant impact on the expression of immune-related genes in the infarcted myocardium. This confirms the immunomodulatory potential of intrapericardially delivered secretomes and opens new therapeutic perspectives in myocardial infarction treatment.

A Fibrin Coating Method of Polypropylene Meshes Enables the Adhesion of Menstrual Blood-Derived Mesenchymal Stromal Cells: A New Delivery Strategy for Stem Cell-Based Therapies.

Polypropylene (PP) mesh is well-known as a gold standard of all prosthetic materials of choice for the reinforcement of soft tissues in case of hernia, organ prolapse, and urinary incontinence. The adverse effects that follow surgical mesh implantation remain an unmet medical challenge. Herein, it is outlined a new approach to allow viability and adhesion of human menstrual blood-derived mesenchymal stromal cells (MenSCs) on PP surgical meshes. A multilayered fibrin coating, based on fibrinogen and thrombin from a commercial fibrin sealant, was optimized to guarantee a homogeneous and stratified film on PP mesh. MenSCs were seeded on the optimized fibrin-coated meshes and their adhesion, viability, phenotype, gene expression, and immunomodulatory capacity were fully evaluated. This coating guaranteed MenSC viability, adhesion and did not trigger any change in their stemness and inflammatory profile. Additionally, MenSCs seeded on fibrin-coated meshes significantly decreased CD4+ and CD8+ T cell proliferation, compared to in vitro stimulated lymphocytes (p < 0.0001). Hence, the proposed fibrin coating for PP surgical meshes may allow the local administration of stromal cells and the reduction of the exacerbated inflammatory response following mesh implantation surgery. Reproducible and easy to adapt to other cell types, this method undoubtedly requires a multidisciplinary and translational approach to be improved for future clinical uses.

IFN-Gamma and TNF-Alpha as a Priming Strategy to Enhance the Immunomodulatory Capacity of Secretomes from Menstrual Blood-Derived Stromal Cells.

Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.

Intrapericardial Delivery of APA-Microcapsules as Promising Stem Cell Therapy Carriers in an Experimental Acute Myocardial Infarction Model.

The administration of cardiosphere-derived cells (CDCs) after acute myocardial infarction (AMI) is very promising. CDC encapsulation in alginate-poly-l-lysine-alginate (APA) could increase cell survival and adherence. The intrapericardial (IP) approach potentially achieves high concentrations of the therapeutic agent in the infarcted area. We aimed to evaluate IP therapy using a saline vehicle as a control (CON), a dose of 30 × 106 CDCs (CDCs) or APA microcapsules containing 30 × 106 CDCs (APA-CDCs) at 72 h in a porcine AMI model. Magnetic resonance imaging (MRI) was used to determine the left ventricular ejection fraction (LVEF), infarct size (IS), and indexed end diastolic and systolic volumes (EDVi; ESVi) pre- and 10 weeks post-injection. Programmed electrical stimulation (PES) was performed to test arrhythmia inducibility before euthanasia. Histopathological analysis was carried out afterwards. The IP infusion was successful in all animals. At 10 weeks, MRI revealed significantly higher LVEF in the APA-CDC group compared with CON. No significant differences were observed among groups in IS, EDVi, ESVi, PES and histopathological analyses. In conclusion, the IP injection of CDCs (microencapsulated or not) was feasible and safe 72 h post-AMI in the porcine model. Moreover, CDCs APA encapsulation could have a beneficial effect on cardiac function, reflected by a higher LVEF at 10 weeks.

The epicardial delivery of cardiosphere derived cells or their extracellular vesicles is safe but of limited value in experimental infarction.

The epicardial administration of therapeutics via the pericardial sac offers an attractive route, since it is minimally invasive and carries no risks of coronary embolization. The aim of this study was to assess viability, safety and effectiveness of cardiosphere-derived cells (CDCs), their extracellular vesicles (EVs) or placebo administered via a mini-thoracotomy 72 h after experimental infarction in swine. The epicardial administration was completed successfully in all cases in a surgery time (knife-to-skin) below 30 min. No significant differences between groups were found in cardiac function parameters evaluated using magnetic resonance imaging before therapy and at the end of the study, despite a trend towards improved function in CDC-treated animals. Moreover, infarct size at 10 weeks was smaller in treated animals, albeit not significantly. Arrhythmia inducibility did not differ between groups. Pathological examination showed no differences, nor were there any pericardial adhesions evidenced in any case 10 weeks after surgery. These results show that the epicardial delivery of CDCs or their EVs is safe and technically easy 3 days after experimental myocardial infarction in swine, but it does not appear to have any beneficial effect on cardiac function. Our results do not support clinical translation of these therapies as implemented in this work.

Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices.

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.

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.

Natural killer cells in recurrent miscarriage: An overview.

Recurrent Miscarriage is an early pregnancy complication which affects about 1-3 % of child-bearing couples. The mechanisms involved in the occurrence of recurrent miscarriages are not clearly understood. In the last decade Natural Killer cells have been studied in peripheral blood and uterus in order to determine if there are specific characteristics of Natural Killer cells associated with miscarriage. Different authors have described an increased number of uterine and peripheral blood Natural Killer cells in women with recurrent miscarriages compared to control women. However, its relationship with miscarriage has not been confirmed. In patients with recurrent miscarriage a lack of inhibition of decidua Natural Killer cells can be observed, which leads to a more activated state characterized by higher levels of proinflammatory cytokines. In peripheral blood, it has been also reported a dysfunctional cytokine production by Natural Killer cells, with an increase of interferon-γ levels and a decrease of Interleukin-4. Significant progress has been made in the last decade in understanding the biology of Natural Killer cells, including the identification of new receptors that also contribute to the activation and regulation of Natural Killer cells. In this review, we summarize the current progress in the study of Natural Killer cells in recurrent miscarriage.

Hydrogels for Bioprinting: A Systematic Review of Hydrogels Synthesis, Bioprinting Parameters, and Bioprinted Structures Behavior.

Nowadays, bioprinting is rapidly evolving and hydrogels are a key component for its success. In this sense, synthesis of hydrogels, as well as bioprinting process, and cross-linking of bioinks represent different challenges for the scientific community. A set of unified criteria and a common framework are missing, so multidisciplinary research teams might not efficiently share the advances and limitations of bioprinting. Although multiple combinations of materials and proportions have been used for several applications, it is still unclear the relationship between good printability of hydrogels and better medical/clinical behavior of bioprinted structures. For this reason, a PRISMA methodology was conducted in this review. Thus, 1,774 papers were retrieved from PUBMED, WOS, and SCOPUS databases. After selection, 118 papers were analyzed to extract information about materials, hydrogel synthesis, bioprinting process, and tests performed on bioprinted structures. The aim of this systematic review is to analyze materials used and their influence on the bioprinting parameters that ultimately generate tridimensional structures. Furthermore, a comparison of mechanical and cellular behavior of those bioprinted structures is presented. Finally, some conclusions and recommendations are exposed to improve reproducibility and facilitate a fair comparison of results.

The Immunomodulatory Signature of Extracellular Vesicles From Cardiosphere-Derived Cells: A Proteomic and miRNA Profiling.

Experimental data demonstrated that the regenerative potential and immunomodulatory capacity of cardiosphere-derived cells (CDCs) is mediated by paracrine mechanisms. In this process, extracellular vesicles derived from CDCs (EV-CDCs) are key mediators of their therapeutic effect. Considering the future applicability of these vesicles in human diseases, an accurate preclinical-to-clinical translation is needed, as well as an exhaustive molecular characterization of animal-derived therapeutic products. Based on that, the main goal of this study was to perform a comprehensive characterization of proteins and miRNAs in extracellular vesicles from porcine CDCs as a clinically relevant animal model. The analysis was performed by identification and quantification of proteins and miRNA expression profiles. Our results revealed the presence of clusters of immune-related and cardiac-related molecular biomarkers in EV-CDCs. Additionally, considering that priming stem cells with inflammatory stimuli may increase the therapeutic potential of released vesicles, here we studied the dynamic changes that occur in the extracellular vesicles from IFNγ-primed CDCs. These analyses detected statistically significant changes in several miRNAs and proteins. Notably, the increase in interleukin 6 (IL6) protein, as well as the increase in mir-125b (that targets IL6 receptor) was especially relevant. These results suggest a potential involvement of EV-CDCs in the regulation of the IL6/IL6R axis, with implications in inflammatory-mediated diseases.

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.

Conditioned Serum Enhances the Chondrogenic and Immunomodulatory Behavior of Mesenchymal Stem Cells.

Osteoarthritis is one of the most common chronic health conditions associated with pain and disability. Advanced therapies based on mesenchymal stem cells have become valuable options for the treatment of these pathologies. Conditioned serum (CS, "Orthokine") has been used intra-articularly for osteoarthritic patients. In this work, we hypothesized that the rich content on anti-inflammatory proteins and growth factors of CS may exert a beneficial effect on the biological activity of human adipose-derived mesenchymal stem cells (hAdMSCs). In vitro studies were designed using hAdMSCs cocultured with CS at different concentrations (2.5, 5, and 10%). Chondrogenic differentiation assays and immunomodulatory experiments using in vitro-stimulated lymphocytes were performed. Our results demonstrated that CS significantly enhanced the differentiation of hAdMSCs toward chondrocytes. Moreover, hAdMSCs pre-sensitized with CS reduced the lymphocyte proliferation as well as their differentiation toward activated lymphocytes. These results suggest that in vivo coadministration of CS and hAdMSCs may have a beneficial effect on the therapeutic potential of hAdMSCs. Moreover, these results indicate that intra-articular administration of CS might influence the biological behavior of resident stem cells increasing their chondrogenic differentiation and inherent immunomodulatory activity. To our knowledge, this is the first in vitro study reporting this combination.

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.