Anna V. Naumova

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell–derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell–derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

Clinical imaging in regenerative medicine

In regenerative medicine, clinical imaging is indispensable for characterizing damaged tissue and for measuring the safety and efficacy of therapy. However, the ability to track the fate and function of transplanted cells with current technologies is limited. Exogenous contrast labels such as nanoparticles give a strong signal in the short term but are unreliable long term. Genetically encoded labels are good both short- and long-term in animals, but in the human setting they raise regulatory issues related to the safety of genomic integration and potential immunogenicity of reporter proteins. Imaging studies in brain, heart and islets share a common set of challenges, including developing novel labeling approaches to improve detection thresholds and early delineation of toxicity and function. Key areas for future research include addressing safety concerns associated with genetic labels and developing methods to follow cell survival, differentiation and integration with host tissue. Imaging may bridge the gap between cell therapies and health outcomes by elucidating mechanisms of action through longitudinal monitoring.

Human embryonic stem cell-derived cardiomyocytes engraft but do not alter cardiac remodeling after chronic infarction in rats

Previous studies indicated that, in an acute myocardial infarction model, human embryonic stem cell-derived cardiomyocytes (hESC-CM) injected with a pro-survival cocktail (PSC) can preserve contractile function. Because patients with established heart failure may also benefit from cell transplantation, we evaluated the physiological effects of hESC-CM transplanted into a chronic model of myocardial infarction. Intramyocardial injection of hESC-CM with PSC was performed in nude rats at 1 month following ischemia-reperfusion. The left ventricular function of hESC-CM injected rats was evaluated at 1, 2 and 3 months after the cell injection procedure and was compared to 3 control groups (rats injected with serum-free media, PSC only, or non-cardiac human cells in PSC). Histology at 3 months revealed that human cardiomyocytes survive, develop increased sarcomere organization and are still proliferating. Despite successful engraftment, both echocardiography and MRI analyses showed no significant difference in left ventricular structure or function between these 4 groups at any time point of the study, suggesting that human cardiomyocytes do not affect cardiac remodeling in a rat model of chronic myocardial infarction. When injected into a chronic infarct model, hESC-CM can engraft, survive and form grafts with striated cardiomyocytes at least as well as was previously observed in an acute myocardial infarction model. However, although hESC-CM transplantation can attenuate the progression of heart failure in an acute model, the same hESC-CM injection protocol is insufficient to restore heart function or to alter adverse remodeling of a chronic myocardial infarction model.

Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart.

An unmet need in cardiac cell therapy is a noninvasive imaging technique capable of tracking changes in graft size over time and monitoring cell dynamics such as replication and death, factors to which commonly used superparamagnetic nanoparticles are insensitive. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart. Mouse skeletal myoblasts (C2C12 cells) were engineered to overexpress ferritin. Ferritin overexpression did not interfere with cell viability, proliferation, or differentiation into multinucleated myotubes. Ferritin overexpression caused a 25% decrease in T2 relaxation time in vitro compared to wild-type cells. Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Graft size evaluation by MRI correlated tighly with histologic measurements (R2 = .8). Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart. These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart.

Quantification of MRI signal of transgenic grafts overexpressing ferritin in murine myocardial infarcts

The noninvasive detection of transplanted cells in damaged organs and the longitudinal follow‐up of cell fate and graft size are important for the evaluation of cell therapy. We have shown previously that the overexpression of the natural iron storage protein, ferritin, permits the detection of engrafted cells in mouse heart by MRI, but further imaging optimization is required. Here, we report a systematic evaluation of ferritin‐based stem cell imaging in infarcted mouse hearts in vivo using three cardiac‐gated pulse sequences in a 3‐T scanner: black‐blood proton‐density‐weighted turbo spin echo (PD TSE BB), bright‐blood T2*‐weighted gradient echo (GRE) and black‐blood T2*‐weighted GRE with improved motion‐sensitized‐driven equilibrium (iMSDE) preparation. Transgenic C2C12 myoblast grafts overexpressing ferritin did not change MRI contrast in the PD TSE BB images, but showed a 20% reduction in signal intensity ratio in black‐blood T2*‐weighted iMSDE (p < 0.05) and a 30% reduction in bright‐blood T2*‐weighted GRE (p < 0.0001). Graft size measurements by T2* iMSDE and T2* GRE were highly correlated with histological assessments (r = 0.79 and r = 0.89, respectively). Unlabeled wild‐type C2C12 cells transplanted to mouse heart did not change the MRI signal intensity, although endogenous hemosiderin was seen in some infarcts. These data support the use of ferritin to track the survival, growth and migration of stem cells transplanted into the injured heart. Copyright

High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields

ABSTRACT A well‐known problem in ultra‐high‐field MRI is generation of high‐resolution three‐dimensional images for detailed characterization of white and gray matter anatomical structures. T1‐weighted imaging traditionally used for this purpose suffers from the loss of contrast between white and gray matter with an increase of magnetic field strength. Macromolecular proton fraction (MPF) mapping is a new method potentially capable to mitigate this problem due to strong myelin‐based contrast and independence of this parameter of field strength. MPF is a key parameter determining the magnetization transfer effect in tissues and defined within the two‐pool model as a relative amount of macromolecular protons involved into magnetization exchange with water protons. The objectives of this study were to characterize the two‐pool model parameters in brain tissues in ultra‐high magnetic fields and introduce fast high‐field 3D MPF mapping as both anatomical and quantitative neuroimaging modality for small animal applications. In vivo imaging data were obtained from four adult male rats using an 11.7 T animal MRI scanner. Comprehensive comparison of brain tissue contrast was performed for standard R1 and T2 maps and reconstructed from Z‐spectroscopic images two‐pool model parameter maps including MPF, cross‐relaxation rate constant, and T2 of pools. Additionally, high‐resolution whole‐brain 3D MPF maps were obtained with isotropic 170 &mgr;m voxel size using the single‐point synthetic‐reference method. MPF maps showed 3–6‐fold increase in contrast between white and gray matter compared to other parameters. MPF measurements by the single‐point synthetic reference method were in excellent agreement with the Z‐spectroscopic method. MPF values in rat brain structures at 11.7 T were similar to those at lower field strengths, thus confirming field independence of MPF. 3D MPF mapping provides a useful tool for neuroimaging in ultra‐high magnetic fields enabling both quantitative tissue characterization based on the myelin content and high‐resolution neuroanatomical visualization with high contrast between white and gray matter. HIGHLIGHTSMacromolecular proton fraction (MPF) mapping was used to image rat brain at 11.7 T.MPF provided the effective source of ultra‐high‐field brain tissue contrast.Fast single‐point MPF mapping method was validated for high‐field MRI applications.MPF mapping enables high‐resolution and high‐contrast imaging of brain anatomy.

Magnetic Resonance Imaging Tracking of Graft Survival in the Infarcted Heart: Iron Oxide Particles Versus Ferritin Overexpression Approach.

The main objective of cell therapy is regeneration of damaged tissues. To distinguish graft from host tissue by MRI, a paramagnetic label must be introduced to cells prior to transplantation. The paramagnetic label can be either exogenous iron oxide nanoparticles or a genetic overexpression of ferritin, an endogenous iron storage protein. The purpose of this work was to compare efficacy of these two methods for MRI evaluation of engrafted cell survival in the infarcted mouse heart. Mouse skeletal myoblasts were labeled either by co-cultivation with iron oxide particles or by engineering them to overexpress ferritin. Along with live cell transplantation, two other groups of mice were injected with dead labeled cells. Both particle-labeled and ferritin-tagged grafts were detected as areas of MRI signal hypointensity in the left ventricle of the mouse heart using T2*-weighted sequences, although the signal attenuation decreased with ferritin tagging. Importantly, live cells could not be distinguished from dead cells when labeled with iron oxide particles, whereas the ferritin-tagging was detected only in live grafts, thereby allowing identification of viable grafts using MRI. Thus, iron oxide particles can provide information about initial cell injection success but cannot assess graft viability. On the other hand, genetically based cell-tagging, such as ferritin overexpression, despite having lower signal intensity in comparison with iron oxide particles, is able to identify live transplanted cells.The main objective of cell therapy is the regeneration of damaged tissues. To distinguish graft from host tissue by magnetic resonance imaging (MRI), a paramagnetic label must be introduced to cells prior to transplantation. The paramagnetic label can be either exogenous iron oxide nanoparticles or a genetic overexpression of ferritin, an endogenous iron storage protein. The purpose of this work was to compare the efficacy of these 2 methods for MRI evaluation of engrafted cell survival in the infarcted mouse heart. Mouse skeletal myoblasts were labeled either by cocultivation with iron oxide particles or by engineering them to overexpress ferritin. Along with live cell transplantation, 2 other groups of mice were injected with dead-labeled cells. Both particle-labeled and ferritin-tagged grafts were detected as areas of MRI signal hypointensity in the left ventricle of the mouse heart using T2*-weighted sequences, although the signal attenuation decreased with ferritin tagging. Importantly, live cells could not be distinguished from dead cells when labeled with iron oxide particles, whereas the ferritin tagging was detected only in live grafts, thereby allowing identification of viable grafts using MRI. Thus, iron oxide particles can provide information about initial cell injection success but cannot assess graft viability. On the other hand, genetically based cell tagging, such as ferritin overexpression, despite having lower signal intensity in comparison with iron oxide particles, is able to identify live transplanted cells.

Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates

Pluripotent stem cell–derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell–derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

Response to Cardiac regeneration validated

Naumova et al. reply: We appreciate the comments from Malliaras and Marbán1 describing their work on developing imaging markers of therapeutic efficacy2 that was not mentioned in our review3. Owing to the page limitation, we were not able to cite all the literature published on this topic. We of course agree that magnetic resonance imaging (MRI) is a valuable technology for evaluating the efficacy of cell therapy, heart contractility, myofiber architecture and infarct size, especially in large-animal models. However, validation of imaging markers requires control measurements, especially in the assessment of gadolinium-contrast kinetics. As these measurements were reported only for cell-treated animals2, it remains unclear whether treatment changes the clearance rate in the scar. Decreases in vascular permeability and reduced extravasation after stem cell transplantation have been reported in controlled studies4,5, indicating that there is experimental evidence rather than a mere theoretical concern regarding these potential confounding factors. Nevertheless, we agree that there was a good correlation between scar size as determined by MRI and histology at the end of the study by Malliaras et al.2, suggesting that cellinduced scar shrinkage may indeed be a real phenomenon. The claimed growth of 10–15 g of new myocardium2 was not as well substantiated. Although there was not an increase in myocyte diameter, cell length was not measured and morphometric estimates of cell number were not obtained. Furthermore, although cell cycle activity was increased threefold, the overall rates To the Editor: In a recent review published in Nature Biotechnology, Naumova et al.1 repeat previously articulated concerns2 about the validity of contrast-enhanced magnetic resonance imaging (MRI) in characterizing myocardial regeneration after cell therapy. The authors raise theoretical concerns that cell administration may promote changes in myocardial vasculature (i.e., a decrease in vascular permeability resulting in reduced gadolinium (Gd)-contrast extravasation or an increase in lymphatic drainage leading to accelerated Gd-contrast washout), which would compromise the ability of contrastenhanced MRI to accurately measure changes in scarred and viable myocardium after cell therapy. In addition, Naumova et al.1 point out (correctly) that MRI cannot distinguish between myocardial hypertrophy and hyperplasia; thus, the increase in viable myocardial mass observed in patients treated with cardiosphere-derived cells (CDCs) could result from hypertrophy of pre-existing cardiomyocytes rather than generation of new myocardium. The concerns are not inconsequential, as they tend to undermine the conclusion that therapeutic regeneration occurred in patients treated with CDCs in the randomized Cardiosphere-derived Autologous Stem Cells to Reverse Ventricular Dysfunction (CADUCEUS) trial3,4. We have addressed these concerns experimentally5 in work not cited in Naumova et al.1. Using a porcine model of ischemic cardiomyopathy that was designed to mimic the protocol in the CADUCEUS trial, we provided direct histological confirmation of the validity of MRI measurements of scar size, scar mass and viable mass. Areas classified as either scarred or viable by MRI agreed precisely with the quantification derived from corresponding tissue sections. In addition, we demonstrated that CDC administration does not alter Gd-contrast kinetics (thus excluding the theoretical possibility of reduced contrast extravasation or accelerated contrast washout), and these findings are consistent with the observed lack of changes in vascular density or architecture. Finally, histological measurement of myocyte size excluded myocyte hypertrophy as a contributor to the increase in viable myocardium observed after CDC therapy. In conclusion, we have validated the fidelity of contrast-enhanced MRI to distinguish and accurately quantify scarred and viable myocardium after cell therapy, supporting its utility for assessing dynamic changes in the were quite low (6 proliferative cells per square millimeter). It remains unclear how such a low rate of proliferation could lead to such a large increase in myocardial mass. Considering the heart’s wellknown proclivity to generate polyploid cardiomyocytes without cell division, a more conservative interpretation of the data would seem prudent until more definitive evidence is available. Still, if the heart did undergo replacement of infarct mass by new cardiomyocytes, it would probably manifest the changes the authors reported by MRI. Additionally, if 10–15 g of new myocardium really were generated through cell division, this should be easy to show by conventional histology techniques, such as BrdU pulselabeling or cumulative labeling experiments. We look forward to seeing such studies in the future. In the meantime, we are intrigued by the potency of cardiospherederived cells to effect cardiac repair but remain cautious about their proposed mechanisms of action.

Assessment of Heart Microstructure

Stem cell therapy has undergone a rapid translation from bench research to clinical trials as a promising approach for the regeneration of the injured myocardium.1,2 Magnetic resonance imaging (MRI) plays a pivotal role in the assessment of stem cell therapy efficacy and elucidation of the mechanisms behind therapeutic effects.3 One important aspect of stem cell therapy, however, remains missing: There are currently no noninvasive methods to evaluate the restoration of myocardial tissue microstructure. A study by Sosnovik et al4 published in this issue of Circulation fills this gap and demonstrates the feasibility of evaluating the integrity and spatial organization of myofibers after cell therapy. Article see p 1731 The microstructure of the heart was described histologically >40 years ago in landmark studies by Streeter and Hanna.5 The myoarchitecture of a healthy heart is made up of 3 layers of crossing spiral myofibers. The subendocardium fiber orientation is a right-handed helix, the subepicardium is a left-handed helix, and fibers in the midmyocardium are circumferential.5 This structure allows for maximal contractile force to ensure effective blood pumping. Despite the discovery of the complex cardiac myoarchitecture and its role in heart function, opportunities to study this aspect of the cardiac anatomy noninvasively were not available for several decades. Diffusion tensor imaging (DTI), the first MRI method capable of visualizing cardiac microstructure, was developed in the mid 1990s.6 DTI allows characterization of anisotropic diffusion of water molecules in tissues. Diffusion anisotropy arises from natural barriers, such as cell membranes, and is most prominent in tissues consisting of coherent fiber bundles, such as brain white matter or muscle. The magnetic resonance signal can be sensitized to diffusion by applying sufficiently strong magnetic field gradients, which cause the loss of phase coherence of individual molecular magnetizations in the …