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Background Anthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute–sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification. Objectives The purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial–specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell–derived cardiomyocytes (iCMs) generated from SENECA patients. Methods Patient-specific iCMs were injured with 1 μmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (, Central Illustration

The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Endogenous capability of the post-mitotic human heart holds great promise to restore the injured myocardium. Recent evidence indicates that the extracellular vesicles (EVs) regulate cardiac homeostasis and regeneration. Here, we investigated the molecular mechanism of EVs for self-repair. We isolated EVs from human iPSC-derived cardiomyocytes (iCMs), which were exposed to hypoxic (hEVs) and normoxic conditions (nEVs), and examined their roles in in vitro and in vivo models of cardiac injury. hEV treatment significantly improved the viability of hypoxic iCMs in vitro and cardiac function of severely injured murine myocardium in vivo. Microarray analysis of the EVs revealed significantly enriched expression of the miR-106a-363 cluster (miR cluster) in hEVs vs. nEVs. This miR cluster preserved survival and contractility of hypoxia-injured iCMs and maintained murine left-ventricular (LV) chamber size, improved LV ejection fraction, and reduced myocardial fibrosis of the injured myocardium. RNA-Seq analysis identified Jag1-Notch3-Hes1 as a target intracellular pathway of the miR cluster. Moreover, the study found that the cell cycle activator and cytokinesis genes were significantly up-regulated in the iCMs treated with miR cluster and Notch3 siRNA. Together, these results suggested that the miR cluster in the EVs stimulated cardiomyocyte cell cycle re-entry by repressing Notch3 to induce cell proliferation and augment myocardial self-repair. The miR cluster may represent an effective therapeutic approach for ischemic cardiomyopathy.

Background: The prognosis for high-grade glioma (HGG) remains dismal and extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Methods: Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients during was scored by pathologists and expression levels were compared to that of mouse xenografts. Results: Intratumoral distribution of pan800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 hours post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Conclusion: Tumor specific uptake of pan800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Introduction: Anthracyclines are effective chemotherapeutic agents associated with cardiac toxicity. Anthracycline induced cardiomyopathy (AIC) carries a mortality of 20% at four years. Effective therapies for AIC have not been established. The NIH-sponsored Stem Cell Injection for Cancer Survivors (SENECA) Trial examined the role of allogeneic mesenchymal stem cells (MSC) to treat AIC. To predict the efficacy of MSC in the SENECA patients, we performed an in vitro clinical trial to study the restorative effects of MSC on patient-specific iPSC-derived cardiomyocytes (iCM) by assessing the effects in vitro following doxorubicin (dox) exposure. Hypothesis: MSC secretome will rescue SENECA Trial patient-specific iCM from dox injury. Methods and Results: We generated three SENECA patient-specific iPSC lines. In vitro iCM dox sensitivity was shown to be comparable to published values. iCM were cultured in 1μM dox for 24hrs, treated with MSC or MSC-secretome, and assessed for viability and apoptosis. Co-culture of MSC with iCM using a 1μm trans-well system improved iCM viability by 43% (p-value < 0.005) and reduced apoptosis (p-value = 0.013). To better define the paracrine mechanism, MSC-secretome was divided into microvesicles >200nm (MV) and exosomes in vitro findings. Conclusion: Allogeneic MSC attenuate apoptosis and restore contractility following dox injury in patient-specific iCM. MV and MT-transfer appear to be the putative mechanism. Our findings correlate with the SENECA results, demonstrating proof-of-concept that iPSC-derivatives can be used to predict trial outcomes.

Introduction: Coronary artery disease is a leading cause of death worldwide. Ischemic injury leads to myocardial dysfunction, resulting in heart failure. Exosomes have emerged as a promising therapeutic for restoring the failing heart. Fundamental questions such as cell of origin and molecular cargo for optimal therapeutic effect are areas of intense research. Our lab has shown that the exosomes from bone marrow derived mesenchymal stem cells (MSC-Ex) and induced pluripotent stem cell derived cardiomyocytes (iCM-Ex) both restore injured murine myocardium. These results led us to compare the therapeutic effects of MSC-Ex vs. iCM-Ex in a porcine myocardial ischemia reperfusion (IR) injury model, a step toward predicting efficacy in humans. Hypothesis: iCM-Ex is superior to MSC-Ex in restoring the injured porcine myocardium. Methods and Results: Pigs underwent ischemia reperfusion (IR) injury, consisting of 1 hour percutaneous balloon occlusion of the proximal left anterior descending artery immediately distal to the first septal artery. Following IR injury, 5 x 10 11 exosomes were delivered in ten, 500μL intramyocardial injections using a BioCardio Helix™ catheter. Biplane ventriculography was used to target the peri-infarct region. At 2- and 4-weeks post-infarct, pigs underwent cardiac MRI (cMRI) with ciné, delayed-enhanced (DEMRI) and manganese-enhanced (MEMRI) MRI. Pigs treated with iCM-Ex (n = 5) demonstrated a 41% improvement in left ventricular ejection fraction (LVEF, p = 0.004) and 35% reduction in indexed left ventricular end diastolic volume (p = 0.008) compared to controls while MSC-Ex (N = 5) did not demonstrate significant functional improvement. Furthermore, DEMRI and MEMRI showed a 21% reduction in myocardial scar (p = 0.14) in iCM-Ex treated animals compared to control while MSC-Ex group showed no difference. RNA-seq of the exosomes and transcriptomic analysis of the ex vivo myocardium will delineate the molecular mechanism of action and the putative intracellular pathway. Conclusion: iCM-Ex is superior to MSC-Ex in improving LVEF and reducing myocardial scar formation following ischemic insult. Comparative analysis between iCM-Ex and MSC-Ex is underway to identify the molecular targets that restore the failing heart.

BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using invivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0× 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0× 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24h after treatment. Finally, intramyocardial injection of 1.0× 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics invitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function invivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.

Background Induced pluripotent stem cells and their differentiated cardiomyocytes ( iCM s) have tremendous potential as patient‐specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCM s (iCM‐Ex) can be robustly collected in vitro and injected in lieu of live iCM s as a cell‐free therapy for myocardial infarction. Methods and Results iCM ‐Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM ‐Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC ‐1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real‐time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM ‐Ex measured ≈140 nm and expressed CD 63 and CD 9. iCM and iCM ‐Ex micro RNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM ‐Ex demonstrated significant cardiac improvement post–myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM ‐Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM ‐Ex groups in vivo and in vitro. Conclusions iCM‐Ex improve post–myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell‐free, patient‐specific therapy for ischemic cardiomyopathy.

Alzheimer's disease (AD) is a type of dementia that causes major issues for patients' memory, thinking, and behavior. Despite efforts to advance AD diagnostic and therapeutic tools, AD remains incurable due to its complex and multifactorial nature and lack of effective diagnostics/therapeutics. Nanoparticles (NPs) have demonstrated the potential to overcome the challenges and limitations associated with traditional diagnostics/therapeutics. Nanotechnology is now offering new tools and insights to advance our understanding of AD and eventually may offer new hope to AD patients. Here, we review the key roles of nanotechnologies in the recent literature, in both diagnostic and therapeutic aspects of AD, and discuss how these achievements may improve patient prognosis and quality of life.

Mitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. Extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source may enhance cardiac function following myocardial injury through restoration of myocardial

Background: Induced pluripotent stem cells (iPSCs) and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for myocardial injury (MI). Our previous work showed that the iCMs restore the injured murine myocardium through secretion of paracrine factors, modulating apoptotic pathways to restore the murine peri-infarct region (PIR). Hypothesis: iCM-derived exosomes (iCM-Ex), a major constituent of the iCM secretome, may salvage the injured cardiomyocytes in the PIR. Methods: iCM-Ex were precipitated from iCM supernatant and characterized using various molecular analyses. Immunodeficient mice sustained MIs and received iCMs, iCM-Ex, or PBS control via direct intramyocardial injection into the PIR. Cardiac MRI assessed LV ejection fraction (LVEF) and viability at 2- and 4-week post-injection. iCMs, iCM-Ex, and PIR tissue were isolated for molecular and histological analyses. Results: iCM-Ex measured approximately 142 nm and expressed CD63 and CD9. iCM and iCM-Ex miRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. In vitro iCM apoptosis was increased significantly by hypoxia and exosome biogenesis inhibition while iCM-Ex or rapamycin reduced iCM apoptosis (p Conclusions: iCM-Ex demonstrated similar efficacy as the iCMs in improving post-MI cardiac function by regulating autophagy and apoptosis of hypoxia injured cardiomyocytes. This finding represents the potential of cell-free, patient-specific biologic to treat ischemic cardiomyopathy by stimulation of an endogenous repair mechanism.

Cellular uptake of nanoparticles (NPs) depends on the nature of the nanobio system including the solid nanocomponents ( e. g., physicochemical properties of NPs), nanobio interfaces ( e. g., protein corona composition), and the cellular characteristics ( e. g., cell type). In this study, we document the role of sex in cellular uptake of NPs as an "overlooked" factor in nanobio interface investigations. We demonstrate that cell sex leads to differences in NP uptake between male and female human amniotic stem cells (hAMSCs), with greater uptake by female cells. hAMSCs are one of the earliest sources of somatic stem cells. The experiments were replicated with primary fibroblasts isolated from the salivary gland of adult male and female donors of similar ages, and again the extent of NP uptake was altered by cell sex. However, in contrast to hAMSCs, uptake was greater in male cells. We also found out that female versus male amniotic stem cells exhibited different responses to reprogramming into induced pluripotent stem cells (iPSCs) by the Yamanaka factors. Thus, future studies should consider the effect of sex on the nanobio interactions to optimize clinical translation of NPs and iPSC biology and to help researchers to better design and produce safe and efficient therapeutic sex-specific NPs.

Stem cell therapy is studied for the treatment of ischemic heart disease. Despite high expectations, investigation has yielded mixed results. For further advancement of this field, it is essential to understand the fate of the transplanted stem cells in living subjects. A myriad of tools has been developed to allow for the immediate and longitudinal monitoring of stem cells in vivo. In this review, we outline the most reliable techniques and their implications for cardiac regenerative medicine. Direct (e.g., PET/SPECT, MRI) and indirect labeling (e.g., reporter gene) techniques have existed for decades prior to their use in stem cell imaging. In this review, we describe some of the key developments in the context of stem cell therapy for cardiac ischemia, including new contrast agents (MRI, SPECT) and novel reporter genes (e.g., near-infrared fluorescent protein). Furthermore, we discuss innovative techniques that integrate direct and indirect labeling, such as PET reporter gene systems. Finally, we examine the potential of exosomes, a component of the stem cell secretome, which has recently garnered much attention for its potential in myocardial regeneration, and how they may be imaged in vivo. This review outlines the most reliable techniques for stem cell imaging in cardiac injury animal models, new and notable advancements in the field, and possible directions for cardiac regenerative medicine.

Rationale: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. Objective: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. Methods and Results: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. Conclusions: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.

Background: Atherosclerotic peripheral artery disease affects 8% to 12% of Americans >65 years of age and is associated with a major decline in functional status, increased myocardial infarction and stroke rates, and increased risk of ischemic amputation. Current treatment strategies for claudication have limitations. PACE (Patients With Intermittent Claudication Injected With ALDH Bright Cells) is a National Heart, Lung, and Blood Institute–sponsored, randomized, double-blind, placebo-controlled, phase 2 exploratory clinical trial designed to assess the safety and efficacy of autologous bone marrow–derived aldehyde dehydrogenase bright (ALDHbr) cells in patients with peripheral artery disease and to explore associated claudication physiological mechanisms. Methods: All participants, randomized 1:1 to receive ALDHbr cells or placebo, underwent bone marrow aspiration and isolation of ALDHbr cells, followed by 10 injections into the thigh and calf of the index leg. The coprimary end points were change from baseline to 6 months in peak walking time (PWT), collateral count, peak hyperemic popliteal flow, and capillary perfusion measured by magnetic resonance imaging, as well as safety. Results: A total of 82 patients with claudication and infrainguinal peripheral artery disease were randomized at 9 sites, of whom 78 had analyzable data (57 male, 21 female patients; mean age, 66±9 years). The mean±SEM differences in the change over 6 months between study groups for PWT (0.9±0.8 minutes; 95% confidence interval [CI] −0.6 to 2.5; P =0.238), collateral count (0.9±0.6 arteries; 95% CI, −0.2 to 2.1; P=0.116), peak hyperemic popliteal flow (0.0±0.4 mL/s; 95% CI, −0.8 to 0.8; P =0.978), and capillary perfusion (−0.2±0.6%; 95% CI, −1.3 to 0.9; P=0.752) were not significant. In addition, there were no significant differences for the secondary end points, including quality-of-life measures. There were no adverse safety outcomes. Correlative relationships between magnetic resonance imaging measures and PWT were not significant. A post hoc exploratory analysis suggested that ALDHbr cell administration might be associated with an increase in the number of collateral arteries (1.5±0.7; 95% CI, 0.1–2.9; P =0.047) in participants with completely occluded femoral arteries. Conclusions: ALDHbr cell administration did not improve PWT or magnetic resonance outcomes, and the changes in PWT were not associated with the anatomic or physiological magnetic resonance imaging end points. Future peripheral artery disease cell therapy investigational trial design may be informed by new anatomic and perfusion insights. Clinical Trial Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01774097.

Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for myocardial injury (MI). Our previous work showed that iCMs secrete paracrine factors, modulating apoptotic pathways to restore the murine peri-infarct region (PIR