Exploiting urine-derived induced pluripotent stem cells for advancing precision medicine in cell therapy, disease modeling, and drug testing.

The field of regenerative medicine has witnessed remarkable advancements with the emergence of induced pluripotent stem cells (iPSCs) derived from a variety of sources. Among these, urine-derived induced pluripotent stem cells (u-iPSCs) have garnered substantial attention due to their non-invasive and patient-friendly acquisition method. This review manuscript delves into the potential and application of u-iPSCs in advancing precision medicine, particularly in the realms of drug testing, disease modeling, and cell therapy. U-iPSCs are generated through the reprogramming of somatic cells found in urine samples, offering a unique and renewable source of patient-specific pluripotent cells. Their utility in drug testing has revolutionized the pharmaceutical industry by providing personalized platforms for drug screening, toxicity assessment, and efficacy evaluation. The availability of u-iPSCs with diverse genetic backgrounds facilitates the development of tailored therapeutic approaches, minimizing adverse effects and optimizing treatment outcomes. Furthermore, u-iPSCs have demonstrated remarkable efficacy in disease modeling, allowing researchers to recapitulate patient-specific pathologies in vitro. This not only enhances our understanding of disease mechanisms but also serves as a valuable tool for drug discovery and development. In addition, u-iPSC-based disease models offer a platform for studying rare and genetically complex diseases, often underserved by traditional research methods. The versatility of u-iPSCs extends to cell therapy applications, where they hold immense promise for regenerative medicine. Their potential to differentiate into various cell types, including neurons, cardiomyocytes, and hepatocytes, enables the development of patient-specific cell replacement therapies. This personalized approach can revolutionize the treatment of degenerative diseases, organ failure, and tissue damage by minimizing immune rejection and optimizing therapeutic outcomes. However, several challenges and considerations, such as standardization of reprogramming protocols, genomic stability, and scalability, must be addressed to fully exploit u-iPSCs' potential in precision medicine. In conclusion, this review underscores the transformative impact of u-iPSCs on advancing precision medicine and highlights the future prospects and challenges in harnessing this innovative technology for improved healthcare outcomes.

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling.

BACKGROUND: Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation. METHODS: hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli. RESULTS: Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features-including enhanced Ca2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells. CONCLUSION: AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

Loss of cardiac myosin light chain kinase contributes to contractile dysfunction in right ventricular pressure overload.

Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post-banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR-200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects.

In vivo evaluation of bioprinted cardiac patches composed of cardiac-specific extracellular matrix and progenitor cells in a model of pediatric heart failure.

Pediatric patients with congenital heart defects (CHD) often present with heart failure from increased load on the right ventricle (RV) due to both surgical methods to treat CHD and the disease itself. Patients with RV failure often require transplantation, which is limited due to lack of donor availability and rejection. Previous studies investigating the development and in vitro assessment of a bioprinted cardiac patch composed of cardiac extracellular matrix (cECM) and human c-kit + progenitor cells (hCPCs) showed that the construct has promise in treating cardiac dysfunction. The current study investigates in vivo cardiac outcomes of patch implantation in a rat model of RV failure. Patch parameters including cECM-inclusion and hCPC-inclusion are investigated. Assessments include hCPC retention, RV function, and tissue remodeling (vascularization, hypertrophy, and fibrosis). Animal model evaluation shows that both cell-free and neonatal hCPC-laden cECM-gelatin methacrylate (GelMA) patches improve RV function and tissue remodeling compared to other patch groups and controls. Inclusion of cECM is the most influential parameter driving therapeutic improvements, with or without cell inclusion. This study paves the way for clinical translation in treating pediatric heart failure using bioprinted GelMA-cECM and hCPC-GelMA-cECM patches.

Designing a 3D Printing Based Auxetic Cardiac Patch with hiPSC-CMs for Heart Repair.

Myocardial infarction is one of the largest contributors to cardiovascular disease and reduces the ability of the heart to pump blood. One promising therapeutic approach to address the diminished function is the use of cardiac patches composed of biomaterial substrates and cardiac cells. These patches can be enhanced with the application of an auxetic design, which has a negative Poisson's ratio and can be modified to suit the mechanics of the infarct and surrounding cardiac tissue. Here, we examined multiple auxetic models (orthogonal missing rib and re-entrant honeycomb in two orientations) with tunable mechanical properties as a cardiac patch substrate. Further, we demonstrated that 3D printing based auxetic cardiac patches of varying thicknesses (0.2, 0.4, and 0.6 mm) composed of polycaprolactone and gelatin methacrylate can support induced pluripotent stem cell-derived cardiomyocyte function for 14-day culture. Taken together, this work shows the potential of cellularized auxetic cardiac patches as a suitable tissue engineering approach to treating cardiovascular disease.

Carfilzomib Treatment Causes Molecular and Functional Alterations of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a cell model for drug-induced cytotoxicity in combination with traction force microscopy, functional assessments, high-throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC-CMs. Following the treatment of hiPSC-CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration-dependent increase in carfilzomib-induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC-CMs in 3-dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca2+ transients and reduced integrin-mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC-CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses.

A systems approach using Diversity Outbred mice distinguishes the cardiovascular effects and genetics of circulating GDF11 from those of its homolog, myostatin.

Growth differentiation factor 11 (GDF11) is a member of the TGF-ß protein family that has been implicated in the development of cardiac hypertrophy. While some studies have suggested that systemic GDF11 protects against cardiomyocyte enlargement and left ventricular wall thickening, there remains uncertainty about the true impact of GDF11 and whether its purported effects are actually attributable to its homolog myostatin. This study was conducted to resolve the statistical and genetic relationships among GDF11, myostatin, and cardiac hypertrophy in a mouse model of human genetics, the Diversity Outbred (DO) stock. In the DO population, serum GDF11 concentrations positively correlated with cardiomyocyte cross-sectional area, while circulating myostatin levels were negatively correlated with body weight, heart weight, and left ventricular wall thickness and mass. Genetic analyses revealed that serum GDF11 concentrations are modestly heritable (0.23) and identified a suggestive peak on murine chromosome 3 in close proximity to the gene Hey1, a transcriptional repressor. Bioinformatic analyses located putative binding sites for the HEY1 protein upstream of the Gdf11 gene in the mouse and human genomes. In contrast, serum myostatin concentrations were more heritable (0.57) than GDF11 concentrations, and mapping identified a significant locus near the gene FoxO1, which has binding motifs within the promoter regions of human and mouse myostatin genes. Together, these findings more precisely define the independent cardiovascular effects of GDF11 and myostatin, as well as their distinct regulatory pathways. Hey1 is a compelling candidate for the regulation of GDF11 and will be further evaluated in future studies.

Functional and molecular effects of TNF-α on human iPSC-derived cardiomyocytes.

Proinflammatory molecule tumor necrosis factor alpha (TNF-α) is predominantly elevated in cytokine storm as well as worsening cardiac function. Here we model the molecular and functional effects of TNF-α in cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC). We found that treatment of hiPSC-CMs with TNF-α increased reactive oxygen species (ROS) and caspase 3/7 activity and caused cell death and apoptosis. TNF-α treatment also resulted in dysregulation of cardiomyocyte function with respect to the increased abnormal calcium handling, calcium wave propagation between cells and excitation-contraction coupling. We also uncovered significant changes in gene expression and protein localization caused by TNF-α treatment. Notably, TNF-α treatment altered the expression of ion channels, dysregulated cadherins, and affected the localization of gap-junction protein connexin-43. In addition, TNF-α treatment up-regulated IL-32 (a human specific cytokine, not present in rodents and an inducer of TNF-α) and IL-34 and down-regulated glutamate receptors and cardiomyocyte contractile proteins. These findings provide insights into the molecular and functional consequences from the exposure of human cardiomyocytes to TNF-α. Our study provides a model to incorporate inflammatory factors into hiPSC-CM-based studies to evaluate mechanistic aspects of heart disease.

Melphalan induces cardiotoxicity through oxidative stress in cardiomyocytes derived from human induced pluripotent stem cells.

BACKGROUND: Treatment-induced cardiotoxicity is a leading noncancer-related cause of acute and late onset morbidity and mortality in cancer patients on antineoplastic drugs such as melphalan-increasing clinical case reports have documented that it could induce cardiotoxicity including severe arrhythmias and heart failure. As the mechanism by which melphalan impairs cardiac cells remains poorly understood, here, we aimed to use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to investigate the cellular and molecular mechanisms of melphalan-induced cardiotoxicity. METHODS: hiPSC-CMs were generated and treated with clinically relevant doses of melphalan. To characterize melphalan-induced cardiotoxicity, cell viability and apoptosis were quantified at various treatment durations. Ca2+ transient and contractility analyses were used to examine the alterations of hiPSC-CM function. Proteomic analysis, reactive oxygen species detection, and RNA-Sequencing were conducted to investigate underlying mechanisms. RESULTS: Melphalan treatment of hiPSC-CMs induced oxidative stress, caused Ca2+ handling defects and dysfunctional contractility, altered global transcriptomic and proteomic profiles, and resulted in apoptosis and cell death. The antioxidant N-acetyl-L-cysteine attenuated these genomic, cellular, and functional alterations. In addition, several other signaling pathways including the p53 and transforming growth factor-ß signaling pathways were also implicated in melphalan-induced cardiotoxicity according to the proteomic and transcriptomic analyses. CONCLUSIONS: Melphalan induces cardiotoxicity through the oxidative stress pathway. This study provides a unique resource of the global transcriptomic and proteomic datasets for melphalan-induced cardiotoxicity and can potentially open up new clinical mechanism-based targets to prevent and treat melphalan-induced cardiotoxicity.

Machine learning identifies abnormal Ca2+ transients in human induced pluripotent stem cell-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an excellent platform for potential clinical and research applications. Identifying abnormal Ca2+ transients is crucial for evaluating cardiomyocyte function that requires labor-intensive manual effort. Therefore, we develop an analytical pipeline for automatic assessment of Ca2+ transient abnormality, by employing advanced machine learning methods together with an Analytical Algorithm. First, we adapt an existing Analytical Algorithm to identify Ca2+ transient peaks and determine peak abnormality based on quantified peak characteristics. Second, we train a peak-level Support Vector Machine (SVM) classifier by using human-expert assessment of peak abnormality as outcome and profiled peak variables as predictive features. Third, we train another cell-level SVM classifier by using human-expert assessment of cell abnormality as outcome and quantified cell-level variables as predictive features. This cell-level SVM classifier can be used to assess additional Ca2+ transient signals. By applying this pipeline to our Ca2+ transient data, we trained a cell-level SVM classifier using 200 cells as training data, then tested its accuracy in an independent dataset of 54 cells. As a result, we obtained 88% training accuracy and 87% test accuracy. Further, we provide a free R package to implement our pipeline for high-throughput CM Ca2+ analysis.

Electrical Stimulation of pediatric cardiac-derived c-kit+ progenitor cells improves retention and cardiac function in right ventricular heart failure.

Nearly 1 in every 120 children born has a congenital heart defect. Although surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. Although primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Here, we investigate the ability of electrical stimulation (ES) to enhance the retention and therapeutic function of pediatric cardiac-derived c-kit+ progenitor cells (CPCs) in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes and increased in vitro survival and adhesion of cells. Specifically, upregulation of ß1 and ß5 integrins contributed to the increased retention of ES-CPCs. Lastly, we show that ES induces CPCs to release higher levels of pro-reparative factors in vitro. These findings suggest that ES can be used to increase the retention, survival, and therapeutic effect of human c-kit+ progenitor cells and can have implications on a variety of cell-based therapies. Stem Cells 2019;37:1528-1541.

Predicting Functional Responses of Progenitor Cell Exosome Potential with Computational Modeling.

Congenital heart disease can lead to severe right ventricular heart failure (RVHF). We have shown that aggregated c-kit+ progenitor cells (CPCs) can improve RVHF repair, likely due to exosome-mediated effects. Here, we demonstrate that miRNA content from monolayer (2D) and aggregated (3D) CPC exosomes can be related to in vitro angiogenesis and antifibrosis responses using partial least squares regression (PLSR). PLSR reduced the dimensionality of the data set to the top 40 miRNAs with the highest weighted coefficients for the in vitro biological responses. Target pathway analysis of these top 40 miRNAs demonstrated significant fit to cardiac angiogenesis and fibrosis pathways. Although the model was trained on in vitro data, we demonstrate that the model can predict angiogenesis and fibrosis responses to exosome treatment in vivo with a strong correlation with published in vivo responses. These studies demonstrate that PLSR modeling of exosome miRNA content has the potential to inform preclinical trials and predict new promising CPC therapies. Stem Cells Translational Medicine 2019;8:1212-1221.

Cardiac Toxicity From Ethanol Exposure in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Alcohol use prior to and during pregnancy remains a significant societal problem and can lead to developmental fetal abnormalities including compromised myocardia function and increased risk for heart disease later in life. Alcohol-induced cardiac toxicity has traditionally been studied in animal-based models. These models have limitations due to physiological differences from human cardiomyocytes (CMs) and are also not suitable for high-throughput screening. We hypothesized that human-induced pluripotent stem cell-derived CMs (hiPSC-CMs) could serve as a useful tool to study alcohol-induced cardiac defects and/or toxicity. In this study, hiPSC-CMs were treated with ethanol at doses corresponding to the clinically relevant levels of alcohol intoxication. hiPSC-CMs exposed to ethanol showed a dose-dependent increase in cellular damage and decrease in cell viability, corresponding to increased production of reactive oxygen species. Furthermore, ethanol exposure also generated dose-dependent increased irregular Ca2+ transients and contractility in hiPSC-CMs. RNA-seq analysis showed significant alteration in genes belonging to the potassium voltage-gated channel family or solute carrier family, partially explaining the irregular Ca2+ transients and contractility in ethanol-treated hiPSC-CMs. RNA-seq also showed significant upregulation in the expression of genes associated with collagen and extracellular matrix modeling, and downregulation of genes involved in cardiovascular system development and actin filament-based process. These results suggest that hiPSC-CMs can be a novel and physiologically relevant system for the study of alcohol-induced cardiac toxicity.

Aggregation of Child Cardiac Progenitor Cells Into Spheres Activates Notch Signaling and Improves Treatment of Right Ventricular Heart Failure.

RATIONALE: Congenital heart disease can lead to life-threatening right ventricular (RV) heart failure. Results from clinical trials support expanding cardiac progenitor cell (CPC) based therapies. However, our recent data show that CPCs lose function as they age, starting as early as 1 year. OBJECTIVE: To determine whether the aggregation of child (1-5-year-old) CPCs into scaffold-free spheres can improve differentiation by enhancing Notch signaling, a known regulator of CPC fate. We hypothesized that aggregated (3-dimensional [3D]) CPCs will repair RV heart failure better than monolayer (2-dimensional [2D]) CPCs. METHODS AND RESULTS: Spheres were produced with 1500 CPCs each using a microwell array. CPC aggregation significantly increased gene expression of Notch1 compared with 2D CPCs, accompanied by significant upregulation of cardiogenic transcription factors (GATA4, HAND1, MEF2C, NKX2.5, and TBX5) and endothelial markers (CD31, FLK1, FLT1, VWF). Blocking Notch receptor activation with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) diminished these effects. To evaluate the therapeutic improvements of CPC aggregation, RV heart failure was induced in athymic rats by pulmonary artery banding, and cells were implanted into the RV free wall. Echocardiographic measurements 28 days postimplantation showed significantly improved RV function with 3D compared with 2D CPCs. Tracking implanted CPCs via DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)-labeling showed improved retention of 3D CPCs. Transducing 3D CPCs with Notch1-shRNA (short hairpin RNA) did not reduce retention, but significantly reduced RV functional improvements. Histological analyses showed 3D treatment reduced RV fibrosis and increased angiogenesis. Although 3D CPCs formed CD31+ vessel-like cells in vivo, these effects are more likely because of improved 3D CPC exosome function compared with 2D CPC exosomes. CONCLUSIONS: Spherical aggregation improves child CPC function in a Notch-dependent manner. The strong reparative ability of CPC spheres warrants further investigation as a treatment for pediatric heart failure, especially in older children where reparative ability may be reduced.

The mitochondrial calcium uniporter underlies metabolic fuel preference in skeletal muscle.

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.

Stem-Cell-Derived Cardiomyocytes Grow Up: Start Young and Train Harder.

Engineering cardiac tissue that accurately recapitulates adult myocardium is critical for advancing disease modeling, drug screening, and regenerative medicine. Ronaldson-Bouchard et al. report a new strategy for generating cardiac tissues from stem-cell-derived cardiomyocytes that reach a maturation level closer to human adult cardiac structure and function.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.

RATIONALE: Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy. OBJECTIVE: We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury. METHODS AND RESULTS: Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. CONCLUSIONS: CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.