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.

Aging Triggers Mitochondrial Dysfunction in Mice.

Direct analysis of isolated mitochondria from old mice enables a better understanding of heart senescence dysfunction. Despite a well-defined senescent phenotype in cardiomyocytes, the mitochondrial state in aged cardiomyocytes is still unclear. Here, we report data about mitochondrial function in old mice. Isolated cardiomyocytes' mitochondria were obtained by differential centrifugation from old and young mice hearts to perform functional analyses of mitochondrial O2 consumption, transmembrane potential, ROS formation, ATP production, and swelling. Our results show that mitochondria from old mouse hearts have reduced oxygen consumption during the phosphorylative states of complexes I and II. Additionally, these mitochondria produced more ROS and less ATP than those of young hearts. Mitochondria from old hearts also showed a depolarized membrane potential than mitochondria from young hearts and, as expected, a greater electron leak. Our results indicate that mitochondria from senescent cardiomyocytes are less efficient in O2 consumption, generating more ROS and producing less ATP. Furthermore, the phosphorylative state of complexes I and II presents a functional defect, contributing to greater leakage of protons and ROS production that can be harmful to the cell.

Optimizing the Decellularized Porcine Liver Scaffold Protocol.

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

Metabolomic profiling suggests systemic signatures of premature aging induced by Hutchinson-Gilford progeria syndrome.

INTRODUCTION: Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder. HGPS children present a high incidence of cardiovascular complications along with altered metabolic processes and an accelerated aging process. No metabolic biomarker is known and the mechanisms underlying premature aging are not fully understood. OBJECTIVES: The present work aims to evaluate the metabolic alterations in HGPS using high resolution mass spectrometry. METHODS: The present study analyzed plasma from six HGPS patients of both sexes (7.7 ± 1.4 years old; mean ± SD) and eight controls (8.6 ± 2.3 years old) by LC-MS/MS in high-resolution non-targeted metabolomics (Q-Exactive Plus). Targeted metabolomics was used to validate some of the metabolites identified by the non-targeted method in a triple quadrupole (TSQ-Quantiva). RESULTS: We found several endogenous metabolites with statistical differences between control and HGPS children. Multivariate statistical analysis showed a clear separation between groups. Potential novel metabolic biomarkers were identified using the multivariate area under ROC curve (AUROC) based analysis, showing an AUC value higher than 0.80 using only two metabolites, and tending to 1.00 when increasing the number of metabolites in the AUROC model. Taken together, changed metabolic pathways involve sphingolipids, amino acids, and oxidation of fatty acids, among others. CONCLUSION: Our data show significant alterations in cellular energy use and availability, in signal transduction, and lipid metabolites, adding new insights on metabolic alterations associated with premature aging and suggesting novel putative biomarkers.

Cell therapies for Chagas disease.

In this review of cell therapies in Chagas disease, we cover aspects related to the disease, its treatment and world demographics, before proceeding to describe the preclinical and clinical trials performed using cell therapies in the search for an alternative therapy for the most severe and lethal form of this disease, chronic chagasic cardiomyopathy.

AT1 and aldosterone receptors blockade prevents the chronic effect of nandrolone on the exercise-induced cardioprotection in perfused rat heart subjected to ischemia and reperfusion.

PURPOSE: Myocardial tolerance to ischaemia/reperfusion (I/R) injury is improved by exercise training, but this cardioprotection is impaired by the chronic use of anabolic androgenic steroids (AAS). The present study evaluated whether blockade of angiotensin II receptor (AT1-R) with losartan and aldosterone receptor (mineralocorticoid receptor, MR) with spironolactone could prevent the deleterious effect of AAS on the exercise-induced cardioprotection. METHODS AND RESULTS: Male Wistar rats were exercised and treated with either vehicle, nandrolone decanoate (10 mg/kg/week i.m.) or the same dose of nandrolone plus losartan or spironolactone (20 mg/kg/day orally) for 8 weeks. Langendorff-perfused hearts were subjected to I/R and evaluated for the postischaemic recovery of left ventricle (LV) function and infarct size. mRNA and protein expression of angiotensin II type 1 receptor (AT1-R), mineralocorticoid receptor (MR), and KATP channels were determined by reverse-transcriptase polymerase chain reaction and Western blotting. Postischaemic recovery of LV function was better and infarct size was smaller in the exercised rat hearts than in the sedentary rat hearts. Nandrolone impaired the exercise-induced cardioprotection, but this effect was prevented by losartan (AT1-R antagonist) and spironolactone (MR antagonist) treatments. Myocardial AT1-R and MR expression levels were increased, and the expression of the KATP channel subunits SUR2a and Kir6.1 was decreased and Kir6.2 increased in the nandrolone-treated rat hearts. The nandrolone-induced changes of AT1-R, MR, and KATP subunits expression was normalized by the losartan and spironolactone treatments. CONCLUSION: The chronic nandrolone treatment impairs the exercise-induced cardioprotection against ischaemia/reperfusion injury by activating the cardiac renin-angiotensin-aldosterone system and downregulating KATP channel expression.

Levels of circulating anti-muscarinic and anti-adrenergic antibodies and their effect on cardiac arrhythmias and dysautonomia in murine models of Chagas disease.

SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as ß 1 and ß 2 adrenergic (anti-ß 1-AR and anti-ß 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-ß 1-AR Ab are detected only in the chronic phase, while anti-ß 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60 dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.

Advanced cell and gene therapies in cardiology.

We review the evidence for the presence of stem/progenitor cells in the heart and the preclinical and clinical data using diverse cell types for the therapy of cardiac diseases. We highlight the failure of adult stem/progenitor cells to ameliorate heart function in most cardiac diseases, with the possible exception of refractory angina. The use of pluripotent stem cell-derived cardiomyocytes is analysed as a viable alternative therapeutic option but still needs further research at preclinical and clinical stages. We also discuss the use of direct reprogramming of cardiac fibroblasts into cardiomyocytes and the use of extracellular vesicles as therapeutic agents in ischemic and non-ischemic cardiac diseases. Finally, gene therapies and genome editing for the treatment of hereditary cardiac diseases, ablation of genes responsible for atherosclerotic disease, or modulation of gene expression in the heart are discussed.

The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy.

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.

Creating an HLA-homozygous iPS cell bank for the Brazilian population: Challenges and opportunities.

Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.