Laminin Alpha 2 Enhances the Protective Effect of Exosomes on Human iPSC-Derived Cardiomyocytes in an In Vitro Ischemia-Reoxygenation Model.

Ischemic heart disease, a leading cause of death worldwide, manifests clinically as myocardial infarction. Contemporary therapies using mesenchymal stromal cells (MSCs) and their derivative (exosomes, EXOs) were developed to decrease the progression of cell damage during ischemic injury. Laminin alpha 2 (LAMA2) is an important extracellular matrix protein of the heart. Here, we generated MSC-derived exosomes cultivated under LAMA2 coating to enhance human-induced pluripotent stem cell (hiPSC)-cardiomyocyte recognition of LAMA2-EXOs, thus, increasing cell protection during ischemia reoxygenation. We mapped the mRNA content of LAMA2 and gelatin-EXOs and identified 798 genes that were differentially expressed, including genes associated with cardiac muscle development and extracellular matrix organization. Cells were treated with LAMA2-EXOs 2 h before a 4 h ischemia period (1% O2, 5% CO2, glucose-free media). LAMA2-EXOs had a two-fold protective effect compared to non-treatment on plasma membrane integrity and the apoptosis activation pathway; after a 1.5 h recovery period (20% O2, 5% CO2, cardiomyocyte-enriched media), cardiomyocytes treated with LAMA2-EXOs showed faster recovery than did the control group. Although EXOs had a protective effect on endothelial cells, there was no LAMA2-enhanced protection on these cells. This is the first report of LAMA2-EXOs used to treat cardiomyocytes that underwent ischemia-reoxygenation injury. Overall, we showed that membrane-specific EXOs may help improve cardiomyocyte survival in treating ischemic cardiovascular disease.

Mesenchymal Stromal Cell-Based Products: Challenges and Clinical Therapeutic Options.

Mesenchymal stromal cell (MSC)-based advanced therapy medicinal products (ATMPs) are being tried in a vast range of clinical applications. These cells can be isolated from different donor tissues by using several methods, or they can even be derived from induced pluripotent stem cells or embryonic stem cells. However, ATMP heterogeneity may impact product identity and potency, and, consequently, clinical trial outcomes. In this review, we discuss these topics and the need to establish minimal criteria regarding the manufacturing of MSCs so that these innovative therapeutics may be better positioned to contribute to the advancement of regenerative medicine.

Decellularized Extracellular Matrix Powder Accelerates Metabolic Maturation at Early Stages of Cardiac Differentiation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45-500 µm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (approx. 70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Whole Heart Engineering: Advances and Challenges.

Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach - recellularization of a decellularized cadaveric organ scaffold with human cells - is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field's 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ.

Machine learning approach for automatic recognition of tomato-pollinating bees based on their buzzing-sounds.

Bee-mediated pollination greatly increases the size and weight of tomato fruits. Therefore, distinguishing between the local set of bees-those that are efficient pollinators-is essential to improve the economic returns for farmers. To achieve this, it is important to know the identity of the visiting bees. Nevertheless, the traditional taxonomic identification of bees is not an easy task, requiring the participation of experts and the use of specialized equipment. Due to these limitations, the development and implementation of new technologies for the automatic recognition of bees become relevant. Hence, we aim to verify the capacity of Machine Learning (ML) algorithms in recognizing the taxonomic identity of visiting bees to tomato flowers based on the characteristics of their buzzing sounds. We compared the performance of the ML algorithms combined with the Mel Frequency Cepstral Coefficients (MFCC) and with classifications based solely on the fundamental frequency, leading to a direct comparison between the two approaches. In fact, some classifiers powered by the MFCC-especially the SVM-achieved better performance compared to the randomized and sound frequency-based trials. Moreover, the buzzing sounds produced during sonication were more relevant for the taxonomic recognition of bee species than analysis based on flight sounds alone. On the other hand, the ML classifiers performed better in recognizing bees genera based on flight sounds. Despite that, the maximum accuracy obtained here (73.39% by SVM) is still low compared to ML standards. Further studies analyzing larger recording samples, and applying unsupervised learning systems may yield better classification performance. Therefore, ML techniques could be used to automate the taxonomic recognition of flower-visiting bees of the cultivated tomato and other buzz-pollinated crops. This would be an interesting option for farmers and other professionals who have no experience in bee taxonomy but are interested in improving crop yields by increasing pollination.

Chromosomal aberrations after induced pluripotent stem cells reprogramming.

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton's jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton's jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.

Cross-talk between autophagy and KLF2 determines endothelial cell phenotype and microvascular function in acute liver injury.

BACKGROUND & AIMS: The transcription factor Krüppel-like factor 2 (KLF2), inducible by simvastatin, confers endothelial vasoprotection. Considering recent data suggesting activation of autophagy by statins, we aimed to: 1) characterize the relationship between autophagy and KLF2 in the endothelium, 2) assess this relationship in acute liver injury (cold ischemia/reperfusion) and 3) study the effects of modulating KLF2-autophagy in vitro and in vivo. METHODS: Autophagic flux, the vasoprotective KLF2 pathway, cell viability and microvascular function were assessed in endothelial cells and in various pre-clinical models of acute liver injury (cold storage and warm reperfusion). RESULTS: Positive feedback between autophagy and KLF2 was observed in the endothelium: KLF2 inducers, pharmacological (statins, resveratrol, GGTI-298), biomechanical (shear stress) or genetic (adenovirus containing KLF2), caused endothelial KLF2 overexpression through a Rac1-rab7-autophagy dependent mechanism, both in the specialized liver sinusoidal endothelial cells (LSEC) and in human umbilical vein endothelial cells. In turn, KLF2 induction promoted further activation of autophagy. Cold ischemia blunted autophagic flux. Upon reperfusion, LSEC stored in University of Wisconsin solution did not reactivate autophagy, which resulted in autophagosome accumulation probably due to impairment in autophagosome-lysosome fusion, ultimately leading to increased cell death and microvascular dysfunction. Simvastatin pretreatment maintained autophagy (through the upregulation of rab7), resulting in increased KLF2, improved cell viability, and ameliorated hepatic damage and microvascular function. CONCLUSIONS: We herein describe for the first time the complex autophagy-KLF2 relationship, modulating the phenotype and survival of the endothelium. These results help understanding the mechanisms of protection conferred by KLF2-inducers, such as simvastatin, in hepatic vascular disorders. LAY SUMMARY: Autophagy and the transcription factor KLF2 share a common activation pathway in the endothelium, being able to regulate each other. Statins maintain microvascular function through the inhibition of Rac1, which consequently liberates Rab7, activates autophagy and increments the expression of KLF2.

Fructose-1,6-bisphosphate reverts iron-induced phenotype of hepatic stellate cells by chelating ferrous ions.

Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-ß1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-ß1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state.

The effects of freeze/thawing on the function and phenotype of CD4(+) lymphocyte subsets in normal individuals and patients with systemic lupus erythematosus.

Several studies report on lymphocyte phenotypic and functional abnormalities in Systemic Lupus Erythematosus (SLE). Freezing and thawing may alter functional and phenotypic properties of cells. We assessed the effect of the freezing/thawing process (F/T) on Th1 (CD3(+)CD4(+)CCR4(-)CXCR3(+)CCR5(+)), Th2 (CD3(+)CD4(+)CCR5(-)CXCR3(-)CCR4(+)), Th17 (CD3(+)CD4(+)CCR6(+)CD161(+)), and Treg (CD3(+)CD4(+)CD25(high)CD127(-)) cell cultures in healthy controls and SLE patients. F/T was associated with decreased frequency of Th2 and Th17 cells in cultures from SLE patients but not from controls. F/T was also associated with increased frequency of apoptotic cells, as measured by annexin V labeling, in all T cell subtypes analyzed, as well as increased cell proliferation, as measured by Ki-67 labeling, in all cells except Th1 from SLE patients. Thus, F/T can have differentiated effects on T lymphocyte subtypes from SLE patients and controls, and can have significant effects on cell death and proliferation. These findings should be carefully considered when designing and interpreting studies on functional and phenotypic aspects of T lymphocytes in SLE.

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress.

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood-derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood-derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H(2)O(2), which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.

Mesenchymal stem cells decrease splenocytes apoptosis in a sepsis experimental model.

OBJECTIVE AND DESIGN: Mesenchymal stem cells (MSCs) are potent modulators of immune responses. Sepsis is the association of a systemic inflammatory response with an infection. The aim of this study was to test the ability of MSCs derived from adipose tissue, which have immunomodulatory effects, and to inhibit the septic process in an experimental model of mice. METHODS: Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10(6) cells/animal). RESULTS: In the control group, there were no deaths; in the untreated septic group, the mortality rate was 100 % within 26 h; in the septic group treated with MSCs, the mortality rate reached 40 % within 26 h. The group treated with MSCs was able to reduce the markers of tissue damage in the liver and pancreas. The treated group had a reduction of inflammatory markers. Furthermore, the MSCs-treated group was able to inhibit the increase of apoptosis in splenocytes observed in the untreated septic group. CONCLUSIONS: Our data showed that MSCs ameliorated the immune response with decrease of inflammatory cytokines and increase anti-inflammatory IL-10; moreover, inhibited splenocytes apoptosis and, consequently, inhibited tissue damage during sepsis.

(+)-Catechin attenuates activation of hepatic stellate cells.

(+)-Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti-inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)-catechin on hepatic stellate cells. (+)-Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator-activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro-inflammatory cytokines were not influenced by (+)-catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)-catechin can reduce hepatic stellate cell activation.

New Biomarkers for Cardiovascular Disease.

Cardiovascular disease is the leading cause of death and disability worldwide. Early detection and treatment of cardiovascular disease are crucial for patient survival and long-term health. Despite advances in cardiovascular disease biomarkers, the prevalence of cardiovascular disease continues to increase worldwide as the global population ages. To address this problem, novel biomarkers that are more sensitive and specific to cardiovascular diseases must be developed and incorporated into clinical practice. Exosomes are promising biomarkers for cardiovascular disease. These small vesicles are produced and released into body fluids by all cells and carry specific information that can be correlated with disease progression. This article reviews the advantages and limitations of existing biomarkers for cardiovascular disease, such as cardiac troponin and cytokines, and discusses recent evidence suggesting the promise of exosomes as cardiovascular disease biomarkers.

CD4 T lymphocyte subsets display heterogeneous susceptibility to apoptosis induced by serum from patients with systemic lupus erythematosus.

BACKGROUND: Serum from systemic lupus erythematosus (SLE) patients has been shown to induce T-lymphocyte (TL) apoptosis. Given that different cells of the immune system display different sensitivity to apoptosis, we set to evaluate the in vitro effect of SLE serum on regulatory T-cells (Treg), Th17, Th1 and Th2 from SLE patients and healthy controls. METHODS: Peripheral blood mononuclear cells from SLE patients or normal controls were exposed to a pool of sera from SLE patients or normal controls. Annexin V was used to label cells in apoptosis or necrosis. Annexin V-labeled Treg, Th17, Th1 and Th2 cells were determined using flow cytometry. RESULTS: Total CD3 + and CD4 + cells from SLE patients showed higher frequency of spontaneous apoptosis/necrosis, whereas Th1 cells from SLE patients presented reduced spontaneous apoptosis/necrosis rate as compared with cells from controls. Incubation with SLE serum induced increased frequency of apoptotic/necrotic CD3 + , CD4 + and Th2 cells from normal controls or from SLE patients as compared with cultures incubated with normal human serum (NHS) or without human serum at all. Incubation with SLE serum did not increase the apoptosis/necrosis rate in Th1 or Th17 cells. Treg cells from SLE patients were more prone to apoptosis/necrosis induced by SLE serum than Treg cells from normal individuals. Th1, Th2, and Th17 cells presented increased apoptosis rates in cultures without human serum. CONCLUSION: Our findings indicate that the serum of patients with active SLE stimulates apoptosis of CD4 + T cells in general and exhibit differentiated effects on CD4 + T-cell subsets.

Automatic acoustic recognition of pollinating bee species can be highly improved by Deep Learning models accompanied by pre-training and strong data augmentation.

Introduction: Bees capable of performing floral sonication (or buzz-pollination) are among the most effective pollinators of blueberries. However, the quality of pollination provided varies greatly among species visiting the flowers. Consequently, the correct identification of flower visitors becomes indispensable to distinguishing the most efficient pollinators of blueberry. However, taxonomic identification normally depends on microscopic characteristics and the active participation of experts in the decision-making process. Moreover, the many species of bees (20,507 worldwide) and other insects are a challenge for a decreasing number of insect taxonomists. To overcome the limitations of traditional taxonomy, automatic classification systems of insects based on Machine-Learning (ML) have been raised for detecting and distinguishing a wide variety of bioacoustic signals, including bee buzzing sounds. Despite that, classical ML algorithms fed by spectrogram-type data only reached marginal performance for bee ID recognition. On the other hand, emerging systems from Deep Learning (DL), especially Convolutional Neural Networks (CNNs), have provided a substantial boost to classification performance in other audio domains, but have yet to be tested for acoustic bee species recognition tasks. Therefore, we aimed to automatically identify blueberry pollinating bee species based on characteristics of their buzzing sounds using DL algorithms. Methods: We designed CNN models combined with Log Mel-Spectrogram representations and strong data augmentation and compared their performance at recognizing blueberry pollinating bee species with the current state-of-the-art models for automatic recognition of bee species. Results and Discussion: We found that CNN models performed better at assigning bee buzzing sounds to their respective taxa than expected by chance. However, CNN models were highly dependent on acoustic data pre-training and data augmentation to outperform classical ML classifiers in recognizing bee buzzing sounds. Under these conditions, the CNN models could lead to automating the taxonomic recognition of flower-visiting bees of blueberry crops. However, there is still room to improve the performance of CNN models by focusing on recording samples for poorly represented bee species. Automatic acoustic recognition associated with the degree of efficiency of a bee species to pollinate a particular crop would result in a comprehensive and powerful tool for recognizing those that best pollinate and increase fruit yields.

Capsaicin induces de-differentiation of activated hepatic stellate cell.

Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. Activation of PPARγ and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. A number of natural products have been suggested to have antifibrotic effects for the de-activation and de-differentiation of HSCs. The purpose of this study was to investigate the in vitro effects of capsaicin on HSC de-activation and de-differentiation. The results demonstrated that capsaicin induced quiescent phenotype in GRX via PPARγ activation. Significant decrease in COX-2 and type I collagen mRNA expression was observed in the first 24 h of treatment. These events preceded the reduction of TGF-ß1 and total collagen secretion. Thus, capsaicin promoted down-regulation of HSC activation by its antifibrotic and anti-inflammatory actions. These findings demonstrate that capsaicin may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

Antiproliferative effect of catechin in GRX cells.

The phenolic compounds present in cocoa seeds have been studied regarding health benefits, such as antioxidant and anti-inflammatory activities. Fibrosis is a wound healing response that occurs in almost all patients with chronic liver injury. A large number of cytokines and soluble intercellular mediators are related to changes in the behavior and phenotype of the hepatic stellate cell (HSC) that develop a fibrogenic and contractile phenotype leading to the development of fibrosis. The objective of this study was to assess the catechin effect in GRX liver cells in activities such as cell growth and inflammation. The GRX cells treatment with catechin induced a significant decrease in cell growth. This mechanism does not occur by apoptosis or even by autophagy because there were no alterations in expression of caspase 3 and PARP (apoptosis), and LC3 (autophagy). The expression of p27 and p53 proteins, regulators of the cell cycle, showed increased expression, while COX-2 and IL-6 mRNA showed a significant decrease in expression. This study shows that catechin decreases cell growth in GRX cells and, probably, this decrease does not occur by apoptosis or autophagy but through an anti-inflammatory effect and cell cycle arrest. Catechin also significantly decreased the production of TGF-ß by GRX cells, showing a significant antifibrotic effect.

Evaluation of the brain and kidney renin-angiotensin system and oxidative stress in neonatal handled rats.

The aim of this study was to test the hypothesis that the renin-angiotensin system (RAS) components, as well as the oxidative stress system, would respond to early environmental changes. Thus, we have evaluated the effects of neonatal handling on both brain and kidney RAS and oxidative stress. Pups were divided into two groups: nonhandled and handled. The procedure consisted of handling them for 1 min/day in the first 10 days of life. On days 1, 5, and 10, animals were killed by decapitation. Blood samples were collected and the brain and kidneys were removed. Renin, AT(1), and AT(2) mRNA expression were evaluated through RT-PCR. Angiotensin II (ANG II) serum concentration was also measured. An increased ANG II concentration, brain and kidney AT(2) mRNA expression were demonstrated. The kidney mRNA AT(1) expression was decreased. There was also a kidney lipid peroxidation increase and a brain superoxide dismutase and catalase decrease. In conclusion, handling in the neonatal period induces the activation of the angiotensinergic system, as well as modulates its mRNA receptor expression. The oxidative stress balance system seems not to be involved.

Mining the Mesenchymal Stromal Cell Secretome in Patients with Chronic Left Ventricular Dysfunction.

Close examination of the initial results of cardiovascular cell therapy clinical trials indicates the importance of patient-specific differences on outcomes and the need to optimize or customize cell therapies. The fields of regenerative medicine and cell therapy have transitioned from using heterogeneous bone marrow mononuclear cells (BMMNCs) to mesenchymal stromal cells (MSCs), which are believed to elicit benefits through paracrine activity. Here, we examined MSCs from the BMMNCs of heart failure patients enrolled in the FOCUS-CCTRN trial. We sought to identify differences in MSCs between patients who improved and those who declined in heart function, regardless of treatment received. Although we did not observe differences in the cell profile of MSCs between groups, we did find significant differences in the MSC secretome profile between patients who improved or declined. We conclude that "mining" the MSC secretome may provide clues to better understand the impact of patient characteristics on outcomes after cell therapy and this knowledge can inform future cell therapy trials.