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.

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.

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.

Empagliflozin reduces arrhythmogenic effects in rat neonatal and human iPSC-derived cardiomyocytes and improves cytosolic calcium handling at least partially independent of NHE1.

The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 µM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.

Genetic Ablation and Pharmacological Blockade of Bradykinin B1 Receptor Unveiled a Detrimental Role for the Kinin System in Chagas Disease Cardiomyopathy.

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

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.

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.

Stem cell therapies in cardiac diseases: Current status and future possibilities.

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.

Inclusivity and diversity: Integrating international perspectives on stem cell challenges and potential.

Regenerative medicine has great potential. The pace of scientific advance is exciting and the medical opportunities for regeneration and repair may be transformative. However, concerns continue to grow, relating to problems caused both by unscrupulous private clinics offering unregulated therapies based on little or no evidence and by premature regulatory approval on the basis of insufficient scientific rationale and clinical evidence. An initiative by the InterAcademy Partnership convened experts worldwide to identify opportunities and challenges, with a focus on stem cells. This was designed to be inclusive and consensus outputs reflected the diversity of the global research population. Among issues addressed for supporting research and innovation while protecting patients were ethical assessment; pre-clinical and clinical research; regulatory authorization and medicines access; and engagement with patients, policy makers, and the public. The InterAcademy Partnership (IAP) identified options for action for sharing good practice and building collaboration within the scientific community and with other stakeholders worldwide.

Cell-Based Therapies for Heart Failure.

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.

New Cardiomyokine Reduces Myocardial Ischemia/Reperfusion Injury by PI3K-AKT Pathway Via a Putative KDEL-Receptor Binding.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.

Therapy with Cardiomyocytes Derived from Pluripotent Cells in Chronic Chagasic Cardiomyopathy.

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.

Exogenous 10 kDa-Heat Shock Protein Preserves Mitochondrial Function After Hypoxia/Reoxygenation.

Humoral factors released during ischemic preconditioning (IPC) protect the myocardium against ischemia/reperfusion (I/R) injury. We have recently identified 10 kDa-heat shock protein (HSP10) and a fraction of small 5-10 kDa peptides (5-10-sP) in the coronary effluent of IPC-treated hearts and demonstrated their cardioprotective potential. We here used our isolated mitochondria model to characterize the impact of exogenous HSP10 and 5-10-sP on mitochondria function from myocardium subjected to I/R injury. Isolated perfused rat hearts were submitted to 30-min global ischemia and 10-min reperfusion. Before ischemia, isolated hearts were infused with saline or 5-10-sP, with or without a mitochondrial ATP-sensitive-K+-channel blocker (5HD 10 µmol·L-1) or PKC inhibitor (chelerythrine 10 µmol·L-1), before I/R. HSP10 (1 µmol·L-1) was infused into isolated hearts before I/R without blockers. At 10-min reperfusion, the mitochondria were isolated and mitochondrial function was assessed. In a subset of experiments, freshly isolated mitochondria were directly incubated with HSP10 or 5-10-sP with or without 5HD or chelerythrine before in vitro hypoxia/reoxygenation. Infusion of 5-10-sP (n = 5) and HSP10 (n = 5) into isolated hearts before I/R improved mitochondrial ADP-stimulated respiration, ATP production and prevented mitochondrial ROS formation compared to the I/R group (n = 5); this effect was abrogated by 5HD and chelerythrine. In freshly isolated mitochondria with in vitro hypoxia/reoxygenation, HSP10 (n = 16) and 5-10-sP (n = 16) incubation prevented reductions of mitochondrial ADP-stimulated respiration (91.5 ± 5.1 nmol O2/min/mg PTN), ATP production (250.1 ± 9.3 µmol ATP/200µg PTN), and prevented mitochondrial ROS production (219.7 ± 9.0 nmol H2O2/200µg PTN) induced by hypoxia/reoxygenation (n = 12, 51.5 ± 5.0 nmol O2/min/mg PTN; 187 ± 21.7 µmol ATP/200 µg PTN; 339.0 ± 14.3 nmol H2O2/200 µg PTN, p < 0.001, respectively). 5HD reduced the ADP-stimulated respiration in the HSP10 group (65.84 ± 3.3 nmol O2/min/mg PTN), ATP production (193.7 ± 12.1 µmol ATP/200µg PTN) and increased ROS in the 5-10-sP group (274.4 ± 21.7 nmol H2O2/200 µg PTN). Mitochondria are a target of the cardioprotection induced by 5-10-sP and HSP10. This protection is dependent of PKC and mKATP activation. HSP10 can act directly on mitochondria and protects against hypoxia/reoxygenation injury by mKATP activation.

Proteomics in the World of Induced Pluripotent Stem Cells.

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.

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.

Aging-related compensated hypogonadism: Role of metabolomic analysis in physiopathological and therapeutic evaluation.

Aging is a complex process that increases the risk of chronic disease development. Hormonal and metabolic alterations occur with aging, such as androgen activity decrease. Studies aim to understand the role of testosterone replacement therapy (TRT) in males, however biomarkers and the metabolic responses to TRT are not well characterized. Therefore, the present study investigated TRT effect in young adult and aged rats by metabolomics. Male Wistar rats were divided into four groups: adult and adult + testo (6months), old and old + testo (25-27months). TRT animals received daily testosterone propionate (1 mg/kg/subcutaneous). TRT changed the testicular weight index decrease induced by aging but did not change the body weight and liver weight index. Sera were analyzed by liquid chromatograph high resolution mass spectrometry (LCMS/MS). Testosterone was quantified by target LCMS/MS. A total of 126 metabolites were detected with known identification altered by TRT by non-target metabolomics analysis. Multivariate statistics shows that all groups segregated individually after principal component analysis. The treatment with testosterone induced several metabolic alterations in adult and old rats that were summarized by variable importance on projection score, metabolite interaction and pathway analysis. Aging-related hypogonadism induces a pattern of systemic metabolic alterations that can be partially reversed by TRT, however, this treatment in aged rats induces novel alterations in some metabolites that are possible new targets for monitoring in patients submitted to TRT.

Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy.

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.

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.

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells.

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-ß1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.

Cardiosphere-derived cells do not improve cardiac function in rats with cardiac failure.

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.