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Martins de Oliveira ML, Tura BR, Meira Leite M, Melo Dos Santos EJ, Pôrto LC, Pereira LV, Campos de Carvalho AC. Creating an HLA-homozygous iPS cell bank for the Brazilian population: Challenges and opportunities. Stem Cell Reports 2023; 18:1905-1912. [PMID: 37774702 PMCID: PMC10656352 DOI: 10.1016/j.stemcr.2023.09.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Revised: 08/31/2023] [Accepted: 09/01/2023] [Indexed: 10/01/2023] [Imported: 10/01/2023] Open
Abstract
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.
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Monnerat G, Kasai-Brunswick TH, Asensi KD, Silva dos Santos D, Barbosa RAQ, Cristina Paccola Mesquita F, Calvancanti Albuquerque JP, Raphaela PF, Wendt C, Miranda K, Domont GB, Nogueira FCS, Bastos Carvalho A, Campos de Carvalho AC. Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient. Front Physiol 2022; 13:1007418. [PMID: 36505085 PMCID: PMC9726722 DOI: 10.3389/fphys.2022.1007418] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2022] [Accepted: 11/07/2022] [Indexed: 11/24/2022] [Imported: 08/29/2023] Open
Abstract
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.
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Kasai-Brunswick TH, Carvalho AB, Campos de Carvalho AC. Stem cell therapies in cardiac diseases: Current status and future possibilities. World J Stem Cells 2021; 13:1231-1247. [PMID: 34630860 PMCID: PMC8474720 DOI: 10.4252/wjsc.v13.i9.1231] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 07/26/2021] [Accepted: 08/10/2021] [Indexed: 02/06/2023] [Imported: 08/29/2023] Open
Abstract
Cardiovascular diseases represent the world’s leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.
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Biagi D, Fantozzi ET, Campos-Oliveira JC, Naghetini MV, Ribeiro AF, Rodrigues S, Ogusuku I, Vanderlinde R, Christie MLA, Mello DB, de Carvalho ACC, Valadares M, Cruvinel E, Dariolli R. In Situ Maturated Early-Stage Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Improve Cardiac Function by Enhancing Segmental Contraction in Infarcted Rats. J Pers Med 2021; 11:jpm11050374. [PMID: 34064343 PMCID: PMC8147857 DOI: 10.3390/jpm11050374] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Revised: 04/21/2021] [Accepted: 04/30/2021] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
The scant ability of cardiomyocytes to proliferate makes heart regeneration one of the biggest challenges of science. Current therapies do not contemplate heart re-muscularization. In this scenario, stem cell-based approaches have been proposed to overcome this lack of regeneration. We hypothesize that early-stage hiPSC-derived cardiomyocytes (hiPSC-CMs) could enhance the cardiac function of rats after myocardial infarction (MI). Animals were subjected to the permanent occlusion of the left ventricle (LV) anterior descending coronary artery (LAD). Seven days after MI, early-stage hiPSC-CMs were injected intramyocardially. Rats were subjected to echocardiography pre-and post-treatment. Thirty days after the injections were administered, treated rats displayed 6.2% human cardiac grafts, which were characterized molecularly. Left ventricle ejection fraction (LVEF) was improved by 7.8% in cell-injected rats, while placebo controls showed an 18.2% deterioration. Additionally, cell-treated rats displayed a 92% and 56% increase in radial and circumferential strains, respectively. Human cardiac grafts maturate in situ, preserving proliferation with 10% Ki67 and 3% PHH3 positive nuclei. Grafts were perfused by host vasculature with no evidence for immune rejection nor ectopic tissue formations. Our findings support the use of early-stage hiPSC-CMs as an alternative therapy to treat MI. The next steps of preclinical development include efficacy studies in large animals on the path to clinical-grade regenerative therapy targeting human patients.
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Campos de Carvalho AC, Kasai-Brunswick TH, Bastos Carvalho A. Cell-Based Therapies for Heart Failure. Front Pharmacol 2021; 12:641116. [PMID: 33912054 PMCID: PMC8072383 DOI: 10.3389/fphar.2021.641116] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2020] [Accepted: 02/11/2021] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
Heart failure has reached epidemic proportions with the advances in cardiovascular therapies for ischemic heart diseases and the progressive aging of the world population. Efficient pharmacological therapies are available for treating heart failure, but unfortunately, even with optimized therapy, prognosis is often poor. Their last therapeutic option is, therefore, a heart transplantation with limited organ supply and complications related to immunosuppression. In this setting, cell therapies have emerged as an alternative. Many clinical trials have now been performed using different cell types and injection routes. In this perspective, we will analyze the results of such trials and discuss future perspectives for cell therapies as an efficacious treatment of heart failure.
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Brasil GV, Silva dos Santos D, Mendonça EA, Mesquita FCP, Kasai-Brunswick TH, da Cunha ST, Pimentel CF, de Vasconcelos-dos-Santos A, Mendez-Otero R, de Azevedo Filho CF, Goldenberg RCDS, Campos de Carvalho AC. Therapy with Cardiomyocytes Derived from Pluripotent Cells in Chronic Chagasic Cardiomyopathy. Cells 2020; 9:cells9071629. [PMID: 32645832 PMCID: PMC7408395 DOI: 10.3390/cells9071629] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 06/30/2020] [Accepted: 07/02/2020] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
Chagas disease discovered more than a century ago remains an incurable disease. The objective of this work was to investigate the therapeutic potential of cardiomyocytes derived from mouse embryonic stem cells (CM-mESC) in a model of chronic Chagasic cardiomyopathy (CCC). Mouse embryonic stem cells (mESC) were characterized, transduced with luciferase, and submitted to cardiac differentiation. CM-mESC were labeled with superparamagnetic iron oxide particles. To induce CCC, mice were infected with Brazil strain trypomastigotes. At 150 days post-infection (dpi), infected animals were treated with CM-mESC or PBS. Cells were detected by magnetic resonance imaging (MRI) and bioluminescence. Cardiac function was evaluated by MRI and electrocardiogram at 150 and 196 dpi. CCC mice showed significant differences in MRI and ECG parameters compared to non-infected mice. However, no differences were observed in contractile and electrical parameters between cell and PBS injected groups, 45 days after cell transplantation. Cells were detected 24 h after transplantation by MRI. CM-mESC bioluminescence tracking demonstrated over 90% decrease in signal 8 days after treatment. Nevertheless, the Infected + CM-mESC group showed a significant reduction in the percentage of collagen fibers when compared to the Infected + PBS group. In conclusion, CM-mESC therapy was not effective in reversing cardiac functional changes induced by Chagas disease despite some improvement in myocardial fibrosis.
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Irion CI, Martins EL, Christie MLA, de Andrade CBV, de Moraes ACN, Ferreira RP, Pimentel CF, Suhett GD, de Carvalho ACC, Lindoso RS, Vieyra A, Galina A, Goldenberg RCS. Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant. Stem Cells Int 2020; 2020:4327965. [PMID: 32655647 PMCID: PMC7322589 DOI: 10.1155/2020/4327965] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 03/02/2020] [Accepted: 03/18/2020] [Indexed: 02/08/2023] [Imported: 08/29/2023] Open
Abstract
Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.
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Hochman-Mendez C, Pereira de Campos DB, Pinto RS, Mendes BJDS, Rocha GM, Monnerat G, Weissmuller G, Sampaio LC, Carvalho AB, Taylor DA, de Carvalho ACC. Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix. J Tissue Eng 2020; 11:2041731420921482. [PMID: 32742631 PMCID: PMC7375712 DOI: 10.1177/2041731420921482] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2019] [Accepted: 03/27/2020] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.
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Mesquita FCP, Arantes PC, Kasai-Brunswick TH, Araujo DS, Gubert F, Monnerat G, Silva Dos Santos D, Neiman G, Leitão IC, Barbosa RAQ, Coutinho JL, Vaz IM, Dos Santos MN, Borgonovo T, Cruz FES, Miriuka S, Medei EH, Campos de Carvalho AC, Carvalho AB. R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome. Sci Rep 2019; 9:19203. [PMID: 31844156 PMCID: PMC6915575 DOI: 10.1038/s41598-019-55837-w] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Accepted: 12/03/2019] [Indexed: 02/06/2023] [Imported: 08/29/2023] Open
Abstract
Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.
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Lindoso RS, Kasai-Brunswick TH, Monnerat Cahli G, Collino F, Bastos Carvalho A, Campos de Carvalho AC, Vieyra A. Proteomics in the World of Induced Pluripotent Stem Cells. Cells 2019; 8:cells8070703. [PMID: 31336746 PMCID: PMC6678893 DOI: 10.3390/cells8070703] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Revised: 06/24/2019] [Accepted: 06/25/2019] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
Omics approaches have significantly impacted knowledge about molecular signaling pathways driving cell function. Induced pluripotent stem cells (iPSC) have revolutionized the field of biological sciences and proteomics and, in particular, has been instrumental in identifying key elements operating during the maintenance of the pluripotent state and the differentiation process to the diverse cell types that form organisms. This review covers the evolution of conceptual and methodological strategies in proteomics; briefly describes the generation of iPSC from a historical perspective, the state-of-the-art of iPSC-based proteomics; and compares data on the proteome and transcriptome of iPSC to that of embryonic stem cells (ESC). Finally, proteomics of healthy and diseased cells and organoids differentiated from iPSC are analyzed.
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Monnerat G, Evaristo GPC, Evaristo JAM, Dos Santos CGM, Carneiro G, Maciel L, Carvalho VO, Nogueira FCS, Domont GB, Campos de Carvalho AC. Metabolomic profiling suggests systemic signatures of premature aging induced by Hutchinson-Gilford progeria syndrome. Metabolomics 2019; 15:100. [PMID: 31254107 DOI: 10.1007/s11306-019-1558-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Accepted: 06/15/2019] [Indexed: 02/05/2023] [Imported: 08/29/2023]
Abstract
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.
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Silva dos Santos D, Brasil GV, Ramos IPR, Mesquita FCP, Kasai-Brunswick TH, Christie MLA, Cahli GM, Barbosa RAQ, da Cunha ST, Pereira JX, Medei E, Campos de Carvalho AC, Carvalho AB, Goldenberg RCDS. Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy. Stem Cell Res Ther 2018; 9:30. [PMID: 29402309 PMCID: PMC5799903 DOI: 10.1186/s13287-018-0788-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Revised: 01/19/2018] [Accepted: 01/23/2018] [Indexed: 02/08/2023] [Imported: 08/29/2023] Open
Abstract
BACKGROUND Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS CM-mESC transplantation improves cardiac function in mice with DIC.
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Carvalho AB, Goldenberg RCDS, Campos de Carvalho AC. Cell therapies for Chagas disease. Cytotherapy 2017; 19:1339-1349. [PMID: 28887011 DOI: 10.1016/j.jcyt.2017.07.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2017] [Accepted: 07/27/2017] [Indexed: 02/08/2023] [Imported: 08/29/2023]
Abstract
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.
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Kasai-Brunswick TH, Costa ARD, Barbosa RAQ, Farjun B, Mesquita FCP, Silva dos Santos D, Ramos IP, Suhett G, Brasil GV, Cunha STD, Brito JOR, Passipieri JDA, Carvalho AB, Campos de Carvalho AC. Cardiosphere-derived cells do not improve cardiac function in rats with cardiac failure. Stem Cell Res Ther 2017; 8:36. [PMID: 28202059 PMCID: PMC5312520 DOI: 10.1186/s13287-017-0481-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2016] [Revised: 01/10/2017] [Accepted: 01/13/2017] [Indexed: 02/08/2023] [Imported: 08/29/2023] Open
Abstract
BACKGROUND Heart failure represents an important public health issue due to its high costs and growing incidence worldwide. Evidence showing the regenerative potential of postmitotic heart tissue has suggested the existence of endogenous cardiac stem cells in adult hearts. Cardiosphere-derived cells (CDC) constitute a candidate pool of such cardiac stem cells. Previous studies using acute myocardial infarction (MI) models in rodents demonstrated an improvement in cardiac function after cell therapy with CDC. We evaluated the therapeutic potential of CDC 60 days after MI in a rat model. METHODS CDC were obtained from human discarded myocardial tissue and rat hearts by enzymatic digestion with collagenase II. At 10-15 days after isolation, small, round, phase-bright cells (PBCs) appeared on top of the adherent fibroblast-like cells. The PBCs were collected and placed on a nonadherent plate for 2 days, where they formed cardiospheres which were then transferred to adherent plates, giving rise to CDC. These CDC were characterized by flow cytometry. Wistar rats were submitted to MI through permanent occlusion of the anterior descending coronary artery. After 60 days, they were immunosuppressed with cyclosporine A during 10 days. On the third day, infarcted animals were treated with 5 × 105 human CDC (hCDC) or placebo through intramyocardial injection guided by echocardiogram. Another group of animals was treated with rat CDC (rCDC) without immunosuppression. hCDC and rCDC were stably transduced with a viral construct expressing luciferase under control of a constitutive promoter. CDC were then used in a bioluminescence assay. Functional parameters were evaluated by echocardiogram 90 and 120 days after MI and by Langendorff at 120 days. RESULTS CDC had a predominantly mesenchymal phenotype. Cell tracking by bioluminescence demonstrated over 85% decrease in signal at 5-7 days after cell therapy. Cardiac function evaluation by echocardiography showed no differences in ejection fraction, end-diastolic volume, or end-systolic volume between groups receiving human cells, rat cells, or placebo. Hemodynamic analyses and infarct area quantification confirmed that there was no improvement in cardiac remodeling after cell therapy with CDC. CONCLUSION Our study challenges the effectiveness of CDC in post-ischemic heart failure.
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Bagno LL, Carvalho D, Mesquita F, Louzada RA, Andrade B, Kasai-Brunswick TH, Lago VM, Suhet G, Cipitelli D, Werneck-de-Castro JP, Campos-de-Carvalho AC. Sustained IGF-1 Secretion by Adipose-Derived Stem Cells Improves Infarcted Heart Function. Cell Transplant 2016; 25:1609-1622. [PMID: 26624235 DOI: 10.3727/096368915x690215] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
The mechanism by which stem cell-based therapy improves heart function is still unknown, but paracrine mechanisms seem to be involved. Adipose-derived stem cells (ADSCs) secrete several factors, including insulin-like growth factor-1 (IGF-1), which may contribute to myocardial regeneration. Our aim was to investigate whether the overexpression of IGF-1 in ADSCs (IGF-1-ADSCs) improves treatment of chronically infarcted rat hearts. ADSCs were transduced with a lentiviral vector to induce IGF-1 overexpression. IGF-1-ADSCs transcribe100- to 200-fold more IGF-1 mRNA levels compared to nontransduced ADSCs. IGF-1 transduction did not alter ADSC immunophenotypic characteristics even under hypoxic conditions. However, IGF-1-ADSCs proliferate at higher rates and release greater amounts of growth factors such as IGF-1, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) under normoxic and hypoxic conditions. Importantly, IGF-1 secreted by IGF-1-ADSCs is functional given that Akt-1 phosphorylation was remarkably induced in neonatal cardiomyocytes cocultured with IGF-1-ADSCs, and this increase was prevented with phosphatidylinositol 3-kinase (PI3K) inhibitor treatment. Next, we tested IGF-1-ADSCs in a rat myocardial infarction (MI) model. MI was performed by coronary ligation, and 4 weeks after MI, animals received intramyocardial injections of either ADSCs (n = 7), IGF-1-ADSCs (n = 7), or vehicle (n = 7) into the infarcted border zone. Left ventricular function was evaluated by echocardiography before and after 6 weeks of treatment, and left ventricular hemodynamics were assessed 7 weeks after cell injection. Notably, IGF-1-ADSCs improved left ventricular ejection fraction and cardiac contractility index, but did not reduce scar size when compared to the ADSC-treated group. In summary, transplantation of ADSCs transduced with IGF-1 is a superior therapeutic approach to treat MI compared to nontransduced ADSCs, suggesting that gene and cell therapy may bring additional benefits to the treatment of MI.
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Martino H, Brofman P, Greco O, Bueno R, Bodanese L, Clausell N, Maldonado JA, Mill J, Braile D, Moraes J, Silva S, Bozza A, Santos B, Campos de Carvalho A. Multicentre, randomized, double-blind trial of intracoronary autologous mononuclear bone marrow cell injection in non-ischaemic dilated cardiomyopathy (the dilated cardiomyopathy arm of the MiHeart study). Eur Heart J 2015; 36:2898.2-2904. [DOI: 10.1093/eurheartj/ehv477] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 08/29/2023] [Imported: 08/29/2023] Open
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Campos de Carvalho AC, Bastos Carvalho A. Stem Cell-Based Therapies in Chagasic Cardiomyopathy. BIOMED RESEARCH INTERNATIONAL 2015; 2015:436314. [PMID: 26161401 PMCID: PMC4486210 DOI: 10.1155/2015/436314] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 05/27/2015] [Indexed: 02/08/2023] [Imported: 08/29/2023]
Abstract
Chagas disease is caused by Trypanosoma cruzi and can lead to a dilated cardiomyopathy decades after the prime infection by the parasite. As with other dilated cardiomyopathies, conventional pharmacologic therapies are not always effective and as heart failure progresses patients need heart transplantation. Therefore alternative therapies are highly desirable and cell-based therapies have been investigated in preclinical and clinical studies. In this paper we review the main findings of such studies and discuss future directions for stem cell-based therapies in chronic chagasic cardiomyopathy.
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Jasmin, Jelicks LA, Tanowitz HB, Peters VM, Mendez-Otero R, de Carvalho ACC, Spray DC. Molecular imaging, biodistribution and efficacy of mesenchymal bone marrow cell therapy in a mouse model of Chagas disease. Microbes Infect 2014; 16:923-935. [PMID: 25218054 PMCID: PMC4360918 DOI: 10.1016/j.micinf.2014.08.016] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2014] [Revised: 07/17/2014] [Accepted: 08/26/2014] [Indexed: 02/08/2023] [Imported: 08/29/2023]
Abstract
Chagasic cardiomyopathy, resulting from infection with the parasite Trypanosoma cruzi, was discovered more than a century ago and remains an incurable disease. Due to the unique properties of mesenchymal stem cells (MSC) we hypothesized that these cells could have therapeutic potential for chagasic cardiomyopathy. Recently, our group pioneered use of nanoparticle-labeled MSC to correlate migration with its effect in an acute Chagas disease model. We expanded our investigation into a chronic model and performed more comprehensive assays. Infected mice were treated with nanoparticle-labeled MSC and their migration was correlated with alterations in heart morphology, metalloproteinase activity, and expression of several proteins. The vast majority of labeled MSC migrated to liver, lungs and spleen whereas a small number of cells migrated to chagasic hearts. Magnetic resonance imaging demonstrated that MSC therapy reduced heart dilatation. Additionally metalloproteinase activity was higher in heart and other organs of infected mice. Protein expression analyses revealed that connexin 43, laminin γ1, IL-10 and INF-γ were affected by the disease and recovered after cell therapy. Interestingly, MSC therapy led to upregulation of SDF-1 and c-kit in the hearts. The beneficial effect of MSC therapy in Chagas disease is likely due to an indirect action of the cells of the heart, rather than the incorporation of large numbers of stem cells into working myocardium.
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Passipieri JA, Kasai-Brunswick TH, Suhett G, Martins AB, Brasil GV, Campos DB, Rocha NN, Ramos IP, Mello DB, Rodrigues DC, Christie BB, Silva-Mendes BJ, Balduíno A, Sá RM, Lopes LM, Goldenberg RC, Campos de Carvalho AC, Carvalho AB. Improvement of cardiac function by placenta-derived mesenchymal stem cells does not require permanent engraftment and is independent of the insulin signaling pathway. Stem Cell Res Ther 2014; 5:102. [PMID: 25145631 PMCID: PMC4354978 DOI: 10.1186/scrt490] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2014] [Revised: 04/18/2014] [Accepted: 08/08/2014] [Indexed: 02/08/2023] [Imported: 08/29/2023] Open
Abstract
INTRODUCTION The objective of this work was to evaluate the efficacy of placenta-derived mesenchymal stem cell (MSC) therapy in a mouse model of myocardial infarction (MI). Since MSCs can be obtained from two different regions of the human term placenta (chorionic plate or villi), cells obtained from both these regions were compared so that the best candidate for cell therapy could be selected. METHODS For the in vitro studies, chorionic plate MSCs (cp-MSCs) and chorionic villi MSCs (cv-MSCs) were extensively characterized for their genetic stability, clonogenic and differentiation potential, gene expression, and immunophenotype. For the in vivo studies, C57Bl/6 mice were submitted to MI and, after 21 days, received weekly intramyocardial injections of cp-MSCs for 3 weeks. Cells were also stably transduced with a viral construct expressing luciferase, under the control of the murine stem cell virus (MSCV) promoter, and were used in a bioluminescence assay. The expression of genes associated with the insulin signaling pathway was analyzed in the cardiac tissue from cp-MSCs and placebo groups. RESULTS Morphology, differentiation, immunophenotype, and proliferation were quite similar between these cells. However, cp-MSCs had a greater clonogenic potential and higher expression of genes related to cell cycle progression and genome stability. Therefore, we considered that the chorionic plate was preferable to the chorionic villi for the isolation of MSCs. Sixty days after MI, cell-treated mice had a significant increase in ejection fraction and a reduction in end-systolic volume. This improvement was not caused by a reduction in infarct size. In addition, tracking of cp-MSCs transduced with luciferase revealed that cells remained in the heart for 4 days after the first injection but that the survival period was reduced after the second and third injections. Quantitative reverse transcription-polymerase chain reaction revealed similar expression of genes involved in the insulin signaling pathway when comparing cell-treated and placebo groups. CONCLUSIONS Improvement of cardiac function by cp-MSCs did not require permanent engraftment and was not mediated by the insulin signaling pathway.
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Daliry A, Caldas IS, de Figueiredo Diniz L, Torres RM, Talvani A, Bahia MT, de Carvalho ACC. Anti-adrenergic and muscarinic receptor autoantibodies in a canine model of Chagas disease and their modulation by benznidazole. Int J Cardiol 2014; 170:e66-7. [PMID: 24268984 DOI: 10.1016/j.ijcard.2013.11.022] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Accepted: 11/02/2013] [Indexed: 02/05/2023] [Imported: 08/29/2023]
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de Carvalho ACC, Carvalho AB. Turning scar into muscle. World J Cardiol 2012; 4:267-70. [PMID: 23024837 PMCID: PMC3460220 DOI: 10.4330/wjc.v4.i9.267] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2012] [Revised: 08/18/2012] [Accepted: 08/25/2012] [Indexed: 02/06/2023] [Imported: 08/29/2023] Open
Abstract
After the demonstration that somatic cells could be reprogrammed to a pluripotent state, exciting new prospects were opened for the cardiac regeneration field. It did not take long for the development of strategies to convert somatic cells directly into cardiomyocytes. Despite the intrinsic difficulties of cell reprogramming, such as low efficiency, the therapeutic possibilities created by the ability to turn scar into muscle are enormous. Here, we discuss some of the major advances and strategies used in direct cardiac reprogramming and examine discrepancies and concerns that still need to be resolved in the field.
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de Carvalho ACC, Carvalho AB, Mello DB, Goldenberg RCDS. Bone marrow-derived cell therapy in chagasic cardiac disease: a review of pre-clinical and clinical results. Cardiovasc Diagn Ther 2012; 2:213-9. [PMID: 24282718 DOI: 10.3978/j.issn.2223-3652.2012.08.03] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2012] [Accepted: 08/22/2012] [Indexed: 11/14/2022] [Imported: 08/29/2023]
Abstract
Chagas disease is caused by a protozoan parasite Trypanosoma cruzi, which infects people through blood sucking insects. It is endemic in Latin America and the disease is being spread to developed countries as a result of the migration of infected individuals. In its chronic stage, Chagas disease can lead to a severe cardiomyopathy for which there is currently no cure. End-stage patients require heart transplantation, thus demanding new therapeutic modalities. Cell-based therapy has been proposed as an alternative for various forms of heart disease. Here we review the experimental evidence that led to the use of bone marrow-derived cells in putative therapy for chronic chagasic cardiomyopathy in animal models and in clinical trials, discussing the reasons for failure of the translation of results from mice to men.
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Ribeiro Dos Santos R, Rassi S, Feitosa G, Grecco OT, Rassi A, da Cunha AB, de Carvalho VB, Guarita-Souza LC, de Oliveira W, Tura BR, Soares MBP, Campos de Carvalho AC. Cell therapy in Chagas cardiomyopathy (Chagas arm of the multicenter randomized trial of cell therapy in cardiopathies study): a multicenter randomized trial. Circulation 2012; 125:2454-61. [PMID: 22523306 DOI: 10.1161/circulationaha.111.067785] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] [Imported: 08/29/2023]
Abstract
BACKGROUND Previous studies suggested that transplantation of autologous bone marrow-derived mononuclear cells (BMNCs) improves heart function in chronic chagasic cardiomyopathy. We report the results of the first randomized trial of BMNC therapy in chronic chagasic cardiomyopathy. METHODS AND RESULTS Patients 18 to 75 years of age with chronic chagasic cardiomyopathy, New York Heart Association class II to IV heart failure, left ventricular ejection fraction (LVEF) <35, and optimized therapy were randomized to intracoronary injection of autologous BMNCs or placebo. The primary end point was the difference in LVEF from baseline to 6 and 12 months after treatment between groups. Analysis was by intention to treat and powered to detect an absolute between-group difference of 5. Between July 2005 and October 2009, 234 patients were enrolled. Two patients abandoned the study and 49 were excluded because of protocol violation. The remaining 183 patients, 93 in the placebo group and 90 in the BMNC group, had a trimmed mean age of 52.4 years (range, 50.8-54.0 years) and LVEF of 26.1 (range, 25.1-27.1) at baseline. Median number of injected BMNCs was 2.20×10(8) (range, 1.40-3.50×10(8)). Change in LVEF did not differ significantly between treatment groups: trimmed mean change in LVEF at 6 months, 3.0 (1.3-4.8) for BMNCs and 2.5 (0.6-4.5) for placebo (P=0.519); change in LVEF at 12 months, 3.5 (1.5-5.5) for BMNCs and 3.7 (1.5-6.0) for placebo (P=0.850). Left ventricular systolic and diastolic volumes, New York Heart Association functional class, Minnesota quality-of-life questionnaire, brain natriuretic peptide concentrations, and 6-minute walking test did also not differ between groups. CONCLUSION Intracoronary injection of autologous BMNCs does not improve left ventricular function or quality of life in patients with chronic chagasic cardiomyopathy.
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Lachtermacher S, Esporcatte BLB, Fortes FDSDA, Rocha NN, Montalvão F, Costa PC, Belem L, Rabischoffisky A, Faria Neto HCC, Vasconcellos R, Iacobas DA, Iacobas S, Spray DC, Thomas NM, Goldenberg RCS, de Carvalho ACC. Functional and transcriptomic recovery of infarcted mouse myocardium treated with bone marrow mononuclear cells. Stem Cell Rev Rep 2012; 8:251-61. [PMID: 21671060 PMCID: PMC3212608 DOI: 10.1007/s12015-011-9282-2] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] [Imported: 08/29/2023]
Abstract
Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice.
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Fidelis-de-Oliveira P, Werneck-de-Castro JPS, Pinho-Ribeiro V, Shalom BCM, Nascimento-Silva JH, Costa e Souza RH, Cruz IS, Rangel RR, Goldenberg RCS, Campos-de-Carvalho AC. Soluble factors from multipotent mesenchymal stromal cells have antinecrotic effect on cardiomyocytes in vitro and improve cardiac function in infarcted rat hearts. Cell Transplant 2012; 21:1011-21. [PMID: 22305373 DOI: 10.3727/096368911x623916] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] [Imported: 08/29/2023] Open
Abstract
The mechanisms underlying the functional improvement after injection of multipotent mesenchymal stromal cells (MSCs) in infarcted hearts remain incompletely understood. The aim of this study was to investigate if soluble factors secreted by MSCs promote cardioprotection. For this purpose, conditioned medium (CM) was obtained after three passages from MSC cultures submitted to 72 h of conditioning in serum-free DMEM under normoxia (NCM) or hypoxia (HCM) conditions. CM was concentrated 25-fold before use (NCM-25X, concentrated normoxia conditioned medium; HCM-25X, concentrated hypoxia conditioned medium). The in vitro cardioprotection was evaluated in neonatal ventricular cardiomyocytes by quantifying apoptosis after 24 h of serum deprivation associated with hypoxia (1% O(2)) in the absence or presence of NCM and HCM (nonconcentrated and 25-fold concentrated). The in vivo cardioprotection of HCM was tested in a model of myocardial infarction (MI) induced in Wistar male rats by permanent left coronary occlusion. Intramyocardial injection of HCM-25X (n = 14) or nonconditioned DMEM (n = 16) was performed 3 h after coronary occlusion and cardiac function was evaluated 19-21 days after medium injection. Cardiac function was evaluated by electro- and echocardiogram, left ventricular catheterization, and treadmill test. The in vitro results showed that HCM was able to decrease cardiomyocyte necrosis. The in vivo results showed that HCM-25X administered 3 h after AMI was able to promote a significant reduction (35%) in left ventricular end-diastolic pressure and improvement of cardiac contractility (15%) and relaxation (12%). These results suggest that soluble factors released in vitro by MSCs are able to promote cardioprotection in vitro and improve cardiac function in vivo.
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