Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

DNA demethylation dynamics.

The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice.

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.

Bioengineering strategies to accelerate stem cell therapeutics.

Although only a few stem cell-based therapies are currently available to patients, stem cells hold tremendous regenerative potential, and several exciting clinical applications are on the horizon. Biomaterials with tuneable mechanical and biochemical properties can preserve stem cell function in culture, enhance survival of transplanted cells and guide tissue regeneration. Rapid progress with three-dimensional hydrogel culture platforms provides the opportunity to grow patient-specific organoids, and has led to the discovery of drugs that stimulate endogenous tissue-specific stem cells and enabled screens for drugs to treat disease. Therefore, bioengineering technologies are poised to overcome current bottlenecks and revolutionize the field of regenerative medicine.

AP-1 is a temporally regulated dual gatekeeper of reprogramming to pluripotency.

Somatic cell transcription factors are critical to maintaining cellular identity and constitute a barrier to human somatic cell reprogramming; yet a comprehensive understanding of the mechanism of action is lacking. To gain insight, we examined epigenome remodeling at the onset of human nuclear reprogramming by profiling human fibroblasts after fusion with murine embryonic stem cells (ESCs). By assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing we identified enrichment for the activator protein 1 (AP-1) transcription factor c-Jun at regions of early transient accessibility at fibroblast-specific enhancers. Expression of a dominant negative AP-1 mutant (dnAP-1) reduced accessibility and expression of fibroblast genes, overcoming the barrier to reprogramming. Remarkably, efficient reprogramming of human fibroblasts to induced pluripotent stem cells was achieved by transduction with vectors expressing SOX2, KLF4, and inducible dnAP-1, demonstrating that dnAP-1 can substitute for exogenous human OCT4. Mechanistically, we show that the AP-1 component c-Jun has two unexpected temporally distinct functions in human reprogramming: 1) to potentiate fibroblast enhancer accessibility and fibroblast-specific gene expression, and 2) to bind to and repress OCT4 as a complex with MBD3. Our findings highlight AP-1 as a previously unrecognized potent dual gatekeeper of the somatic cell state.

Corrigendum: Dermatologist-level classification of skin cancer with deep neural networks.

This corrects the article DOI: 10.1038/nature21056.

Dermatologist-level classification of skin cancer with deep neural networks.

Skin cancer, the most common human malignancy, is primarily diagnosed visually, beginning with an initial clinical screening and followed potentially by dermoscopic analysis, a biopsy and histopathological examination. Automated classification of skin lesions using images is a challenging task owing to the fine-grained variability in the appearance of skin lesions. Deep convolutional neural networks (CNNs) show potential for general and highly variable tasks across many fine-grained object categories. Here we demonstrate classification of skin lesions using a single CNN, trained end-to-end from images directly, using only pixels and disease labels as inputs. We train a CNN using a dataset of 129,450 clinical images-two orders of magnitude larger than previous datasets-consisting of 2,032 different diseases. We test its performance against 21 board-certified dermatologists on biopsy-proven clinical images with two critical binary classification use cases: keratinocyte carcinomas versus benign seborrheic keratoses; and malignant melanomas versus benign nevi. The first case represents the identification of the most common cancers, the second represents the identification of the deadliest skin cancer. The CNN achieves performance on par with all tested experts across both tasks, demonstrating an artificial intelligence capable of classifying skin cancer with a level of competence comparable to dermatologists. Outfitted with deep neural networks, mobile devices can potentially extend the reach of dermatologists outside of the clinic. It is projected that 6.3 billion smartphone subscriptions will exist by the year 2021 (ref. 13) and can therefore potentially provide low-cost universal access to vital diagnostic care.

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy.

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.

Telomere shortening is a hallmark of genetic cardiomyopathies.

This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: TNNI3, MYBPC3, MYH7, DMD, TNNT2, and TTN Quantitative FISH (Q-FISH) of single cells revealed that telomeres were significantly reduced by 26% in HCM and 40% in DCM patient CMs in fixed tissue sections compared with CMs from age- and sex-matched healthy controls. In the cardiac tissues of the same patients, telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs) measured by two independent assays. This study reveals telomere shortening as a hallmark of genetic HCM and DCM and demonstrates that this shortening can be modeled in vitro by using the hiPSC platform, enabling drug discovery.

An objective comparison of cell-tracking algorithms.

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.

An In Vitro Model for Identifying Cardiac Side Effects of Anesthetics.

The understanding of anesthetic side effects on the heart has been hindered by the lack of sophisticated clinical models. Using micropatterned human-induced pluripotent stem cell-derived cardiomyocytes, we obtained cardiac muscle depressant profiles for propofol, etomidate, and our newly identified anesthetic compound KSEB01-S2. Propofol was the strongest depressant among the 3 compounds tested, exhibiting the largest decrease in contraction velocity, depression rate, and beating frequency. Interestingly, KSEB01-S2 behaved similarly to etomidate, suggesting a better cardiac safety profile. Our results provide a proof-of-concept for using human-induced pluripotent stem cell-derived cardiomyocytes as an in vitro platform for future drug design.

Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation.

Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice.

Prostaglandin E2 is essential for efficacious skeletal muscle stem-cell function, augmenting regeneration and strength.

Skeletal muscles harbor quiescent muscle-specific stem cells (MuSCs) capable of tissue regeneration throughout life. Muscle injury precipitates a complex inflammatory response in which a multiplicity of cell types, cytokines, and growth factors participate. Here we show that Prostaglandin E2 (PGE2) is an inflammatory cytokine that directly targets MuSCs via the EP4 receptor, leading to MuSC expansion. An acute treatment with PGE2 suffices to robustly augment muscle regeneration by either endogenous or transplanted MuSCs. Loss of PGE2 signaling by specific genetic ablation of the EP4 receptor in MuSCs impairs regeneration, leading to decreased muscle force. Inhibition of PGE2 production through nonsteroidal anti-inflammatory drug (NSAID) administration just after injury similarly hinders regeneration and compromises muscle strength. Mechanistically, the PGE2 EP4 interaction causes MuSC expansion by triggering a cAMP/phosphoCREB pathway that activates the proliferation-inducing transcription factor, Nurr1 Our findings reveal that loss of PGE2 signaling to MuSCs during recovery from injury impedes muscle repair and strength. Through such gain- or loss-of-function experiments, we found that PGE2 signaling acts as a rheostat for muscle stem-cell function. Decreased PGE2 signaling due to NSAIDs or increased PGE2 due to exogenous delivery dictates MuSC function, which determines the outcome of regeneration. The markedly enhanced and accelerated repair of damaged muscles following intramuscular delivery of PGE2 suggests a previously unrecognized indication for this therapeutic agent.

Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.

AIMS: Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery. METHODS AND RESULTS: In the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by ß-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload. CONCLUSION: In summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.

Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy.

Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/ß leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

Short telomeres - A hallmark of heritable cardiomyopathies.

Cardiovascular diseases are the leading cause of death worldwide and the incidence increases with age. Genetic testing has taught us much about the pathogenic pathways that drive heritable cardiomyopathies. Here we discuss an unexpected link between shortened telomeres, a molecular marker of aging, and genetic cardiomyopathy. Positioned at the ends of chromosomes, telomeres are DNA repeats which serve as protective caps that shorten with each cell division in proliferative tissues. Cardiomyocytes are an anomaly, as they are largely non-proliferative post-birth and retain relatively stable telomere lengths throughout life in healthy individuals. However, there is mounting evidence that in disease states, cardiomyocyte telomeres significantly shorten. Moreover, this shortening may play an active role in the development of mitochondrial dysfunction central to the etiology of dilated and hypertrophic cardiomyopathies. Elucidation of the mechanisms that underlie the telomere-mitochondrial signaling axis in the heart will provide fresh insights into our understanding of genetic cardiomyopathies, and could lead to the identification of previously uncharacterized modes of therapeutic intervention.

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Objective comparison of particle tracking methods.

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.