Background-Ex vivo expansion of resident cardiac stem cells, followed by delivery to the heart, may favor regeneration and functional improvement. Methods and Results-Percutaneous endomyocardial biopsy specimens grown in primary culture developed multicellular clusters known as cardiospheres, which were plated to yield cardiosphere-derived cells (CDCs). CDCs from human biopsy specimens and from comparable porcine samples were examined in vitro for biophysical and cytochemical evidence of cardiogenic differentiation. In addition, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice. Biopsy specimens from 69 of 70 patients yielded cardiosphere-forming cells. Cardiospheres and CDCs expressed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for cardiac contractile and electrical function. Human and porcine CDCs cocultured with neonatal rat ventricular myocytes exhibited biophysical signatures characteristic of myocytes, including calcium transients synchronous with those of neighboring myocytes. Human CDCs injected into the border zone of myocardial infarcts engrafted and migrated into the infarct zone. After 20 days, the percentage of viable myocardium within the infarct zone was greater in the CDC-treated group than in the fibroblast-treated control group; likewise, left ventricular ejection fraction was higher in the CDC-treated group. Conclusions-A method is presented for the isolation of adult human stem cells from endomyocardial biopsy specimens.CDCs are cardiogenic in vitro; they promote cardiac regeneration and improve heart function in a mouse infarct model, which provides motivation for further development for therapeutic applications in patients. Key Words: cells Ⅲ biopsy Ⅲ electrophysiology Ⅲ myocardial infarction Ⅲ myocytes W e sought to develop a clinically applicable method for the isolation and expansion of adult stem cells capable of regenerating myocytes and vessels and improving function in the injured heart. Given recent evidence that the adult mammalian heart contains endogenous, cardiac-committed stem cells, 1-5 we began with cardiac tissue as our stem cell source, postulating that cardiac-derived cells might be particularly well-suited for myocardial regeneration. Percutaneous endomyocardial biopsy specimens were utilized as a convenient, minimally invasive tissue source. 6,7 We began with the observation that cardiac surgical biopsy specimens in culture yield spherical multicellular clusters dubbed "cardiospheres." 8 Cardiospheres resemble neurospheres 9 in that they are derived from primary tissue culture and contain many proliferative cells that express stem cell-related antigens, as well as other cells undergoing spontaneous cardiac differentiation. 8 We modified the original culture method to improve efficiency and added a postcardiosphere expansion step to obtain reasonable numbers of cells (cardiosphere-derived cells [CDCs]) for transplantation from the small specimens in a timely manner. Editori...

Summary Background Cardiosphere-derived cells (CDCs) reduce scarring after myocardial infarction, increase viable myocardium, and boost cardiac function in preclinical models. We aimed to assess safety of such an approach in patients with left ventricular dysfunction after myocardial infarction. Methods In the prospective, randomised CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial, we enrolled patients 2–4 weeks after myocardial infarction (with left ventricular ejection fraction of 25–45%) at two medical centres in the USA. An independent data coordinating centre randomly allocated patients in a 2:1 ratio to receive CDCs or standard care. For patients assigned to receive CDCs, autologous cells grown from endomyocardial biopsy specimens were infused into the infarct-related artery 1·5–3 months after myocardial infarction. The primary endpoint was proportion of patients at 6 months who died due to ventricular tachycardia, ventricular fibrillation, or sudden unexpected death, or had myocardial infarction after cell infusion, new cardiac tumour formation on MRI, or a major adverse cardiac event (MACE; composite of death and hospital admission for heart failure or non-fatal recurrent myocardial infarction). We also assessed preliminary efficacy endpoints on MRI by 6 months. Data analysers were masked to group assignment. This study is registered with ClinicalTrials.gov, NCT00893360. Findings Between May 5, 2009, and Dec 16, 2010, we randomly allocated 31 eligible participants of whom 25 were included in a per-protocol analysis (17 to CDC group and eight to standard of care). Mean baseline left ventricular ejection fraction (LVEF) was 39% (SD 12) and scar occupied 24% (10) of left ventricular mass. Biopsy samples yielded prescribed cell doses within 36 days (SD 6). No complications were reported within 24 h of CDC infusion. By 6 months, no patients had died, developed cardiac tumours, or MACE in either group. Four patients (24%) in the CDC group had serious adverse events compared with one control (13%; p=1·00). Compared with controls at 6 months, MRI analysis of patients treated with CDCs showed reductions in scar mass (p=0·001), increases in viable heart mass (p=0·01) and regional contractility (p=0·02), and regional systolic wall thickening (p=0·015). However, changes in end-diastolic volume, end-systolic volume, and LVEF did not differ between groups by 6 months. Interpretation We show intracoronary infusion of autologous CDCs after myocardial infarction is safe, warranting the expansion of such therapy to phase 2 study. The unprecedented increases we noted in viable myocardium, which are consistent with therapeutic regeneration, merit further assessment of clinical outcomes. Funding US National Heart, Lung and Blood Institute and Cedars-Sinai Board of Governors Heart Stem Cell Center.

SummaryThe CADUCEUS trial of cardiosphere-derived cells (CDCs) has shown that it may be possible to regenerate injured heart muscle previously thought to be permanently scarred. The mechanisms of benefit are known to be indirect, but the mediators have yet to be identified. Here we pinpoint exosomes secreted by human CDCs as critical agents of regeneration and cardioprotection. CDC exosomes inhibit apoptosis and promote proliferation of cardiomyocytes, while enhancing angiogenesis. Injection of exosomes into injured mouse hearts recapitulates the regenerative and functional effects produced by CDC transplantation, whereas inhibition of exosome production by CDCs blocks those benefits. CDC exosomes contain a distinctive complement of microRNAs, with particular enrichment of miR-146a. Selective administration of a miR-146a mimic reproduces some (but not all) of the benefits of CDC exosomes. The findings identify exosomes as key mediators of CDC-induced regeneration, while highlighting the potential utility of exosomes as cell-free therapeutic candidates.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level.

Reactive oxygen species (ROS) and/or Ca 2؉ overload can trigger depolarization of mitochondrial inner membrane potential (⌬⌿ m ) and cell injury. Little is known about how loss of ⌬⌿ m in a small number of mitochondria might influence the overall function of the cell. Here we employ the narrow focal excitation volume of the two-photon microscope to examine the effect of local mitochondrial depolarization in guinea pig ventricular myocytes. Remarkably, a single local laser flash triggered synchronized and self-sustained oscillations in ⌬⌿ m , NADH, and ROS after a delay of ϳ40s, in more than 70% of the mitochondrial population. Oscillations were initiated only after a specific threshold level of mitochondrially produced ROS was exceeded, and did not involve the classical permeability transition pore or intracellular Ca 2؉ overload. The synchronized transitions were abolished by several respiratory inhibitors or a superoxide dismutase mimetic. Anion channel inhibitors potentiated matrix ROS accumulation in the flashed region, but blocked propagation to the rest of the myocyte, suggesting that an inner membrane, superoxide-permeable, anion channel opens in response to free radicals. The transitions in mitochondrial energetics were tightly coupled to activation of sarcolemmal K ATP currents, causing oscillations in action potential duration, and thus might contribute to catastrophic arrhythmias during ischemia-reperfusion injury.Mitochondria play a multifunctional role as key arbiters of cell life and death. In addition to oxidative phosphorylation, mitochondria are involved in thermogenesis, free radical production, and intracellular Ca 2ϩ homeostasis. Impairment of mitochondrial function during and after ischemia causes rapid energy depletion, contractile failure, and loss of cellular integrity, which may lead to necrotic or apoptotic cell death (1-3). ROS 1 have been implicated in ischemic dysfunction; however, they play a dual role as determinants of cell survival, on the one hand contributing to Ca 2ϩ overload and the induction of a mitochondrial permeability transition, but on the other acting as second messengers that protect cells against injury (4 -6). Mitochondria are a major site of physiological ROS production in the cardiomyocyte, with ϳ1-5% of the electrons flowing through the electron transport chain leaking into the production of ROS (7, 8). The negative effects of ROS on metabolism are evident in several studies showing rapid and spatiotemporally heterogeneous discharge of ⌬⌿ m in response to oxidative stress (1, 2) and/or Ca 2ϩ overload (9), including protocols employing laser-induced photooxidation (10, 11) and mitochondrial ROS-induced ROS release (12). In light of our previous work showing that substrate deprivation can initiate synchronized oscillations of mitochondrial redox and membrane potential (13), and that a diffusible cytoplasmic messenger may be involved (14), the present study tests whether similar global self-organizing behavior can be triggered by a highly localized perturbation of ...