Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model of Duchenne Muscular Dystrophy.

Cell therapy for muscular dystrophy has met with limited success, mainly due to the poor engraftment of donor cells, especially in fibrotic muscle at an advanced stage of the disease. We developed a cell-mediated exon skipping that exploits the multinucleated nature of myofibers to achieve cross-correction of resident, dystrophic nuclei by the U7 small nuclear RNA engineered to skip exon 51 of the dystrophin gene. We observed that co-culture of genetically corrected human DMD myogenic cells (but not of WT cells) with their dystrophic counterparts at a ratio of either 1:10 or 1:30 leads to dystrophin production at a level several folds higher than what predicted by simple dilution. This is due to diffusion of U7 snRNA to neighbouring dystrophic resident nuclei. When transplanted into NSG-mdx-Δ51mice carrying a mutation of exon 51, genetically corrected human myogenic cells produce dystrophin at much higher level than WT cells, well in the therapeutic range, and lead to force recovery even with an engraftment of only 3-5%. This level of dystrophin production is an important step towards clinical efficacy for cell therapy.

Nonlinear fiber effects in ultra-high power 10 × 10 Gbit/s WDM-IM free-space systems for satellite links.

We investigate an unexplored type of nonlinear impairments that will take place in a very short fiber after the booster amplifier in a Free Space Optical (FSO) system for space communications. In Earth-satellite links, optical power levels up to 100 W could be required at the transmitter side to achieve the foreseen 100 Gbit/s capacity, because of the extremely high losses. These systems thus need an optical booster amplifier having very high optical power and it should be connected to the transmitting telescope by means of a short fiber (few meters). Here, we discuss and investigate the impact of the nonlinear fiber effects by means of numerical simulations, and estimate the impairments in a Wavelength Division Multiplexing (WDM) 10 × 10 Gbit/s system with intensity modulation. The obtained results clearly indicate that, in this system, the most relevant effect is Four Wave Mixing. We proved that this can be observed as soon as the total power exceeds 20 W. Due to the short fiber length, the system impairments are not affected by chromatic dispersion or channel spacing. We demonstrate that an effective means to reduce the impact is by adopting Polarization Interleaving, i.e., odd and even channels with orthogonal state of polarization. This solution could not work in long terrestrial links because of polarization mode dispersion, yet it can be effectively exploited in short fiber patch cords. These results can be used as a guideline to control this type of impairment in high-power FSO systems for satellite links.

Potency assays and biomarkers for cell-based advanced therapy medicinal products.

Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro, with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo. Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs.

Cost and availability of novel cell and gene therapies: Can we avoid a catastrophic second valley of death?: Can we avoid a catastrophic second valley of death?

Advanced gene and cellular therapies risk a second "valley of death" due to their high costs and low patient population. As these are life-saving therapies, measures are urgently needed to prevent their withdrawal from the market.

The Transcriptional Regulator Prdm1 Is Essential for the Early Development of the Sensory Whisker Follicle and Is Linked to the Beta-Catenin First Dermal Signal.

Prdm1 mutant mice are one of the rare mutant strains that do not develop whisker hair follicles while still displaying a pelage. Here, we show that Prdm1 is expressed at the earliest stage of whisker development in clusters of mesenchymal cells before placode formation. Its conditional knockout in the murine soma leads to the loss of expression of Bmp2, Shh, Bmp4, Krt17, Edar, and Gli1, though leaving the ß-catenin-driven first dermal signal intact. Furthermore, we show that Prdm1 expressing cells not only act as a signaling center but also as a multipotent progenitor population contributing to the several lineages of the adult whisker. We confirm by genetic ablation experiments that the absence of macro vibrissae reverberates on the organization of nerve wiring in the mystacial pads and leads to the reorganization of the barrel cortex. We demonstrate that Lef1 acts upstream of Prdm1 and identify a primate-specific deletion of a Lef1 enhancer named Leaf. This loss may have been significant in the evolutionary process, leading to the progressive defunctionalization and disappearance of vibrissae in primates.

The contracture-in-a-well. An in vitro model distinguishes bulk and interfacial processes of irreversible (fibrotic) cell-mediated contraction.

Tissue contractures are processes of cell-mediated contraction, irreversible in nature and typically associated with fibrotic phenomena. Contractures can be reproduced in vitro; here, we have used a medium-throughput model based on fibroblast-seeded fibrin (the 'contracture well'). Firstly, we show how profoundly these processes depend on the location of the contractile cells: when on top of the material, fibroblasts produce an interfacial contracture (analog to capsular contraction around an implant), which tries and bends the construct; when seeded inside the material, they initiate a bulk contracture (analogue to a wound bed closure) that shrinks it from within. Secondly, we demonstrate that the interfacial and bulk contractures are also mechanically and biologically different processes. Thirdly, we show the potentially predictive value of this model, since it not only recapitulates the effect of pro-fibrotic factors (TGF-ß1 for dermal (myo)fibroblasts), but can also indicate the fibrotic potential of a given cell population (here, dystrophic myoblasts more fibrotic than healthy or genetically corrected ones), which may have important implications in the identification of appropriate therapies.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Challenges in cell transplantation for muscular dystrophy.

For decades now, cell transplantation has been considered a possible therapeutic strategy for muscular dystrophy, but failures have largely outnumbered success or at least encouraging outcomes. In this review we will briefly recall the history of cell transplantation, discuss the peculiar features of skeletal muscle, and dystrophic skeletal muscle in particular, that make the procedure complicated and inefficient. As there are many recent and exhaustive reviews on the various myogenic cell types that have been or will be transplanted, we will only briefly describe them and refer the reader to these reviews. Finally, we will discuss possible strategies to overcome the hurdles that prevent biological efficacy and hence clinical success.

Myogenic Cell Transplantation in Genetic and Acquired Diseases of Skeletal Muscle.

This article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies.

Inclusivity and diversity: Integrating international perspectives on stem cell challenges and potential.

Regenerative medicine has great potential. The pace of scientific advance is exciting and the medical opportunities for regeneration and repair may be transformative. However, concerns continue to grow, relating to problems caused both by unscrupulous private clinics offering unregulated therapies based on little or no evidence and by premature regulatory approval on the basis of insufficient scientific rationale and clinical evidence. An initiative by the InterAcademy Partnership convened experts worldwide to identify opportunities and challenges, with a focus on stem cells. This was designed to be inclusive and consensus outputs reflected the diversity of the global research population. Among issues addressed for supporting research and innovation while protecting patients were ethical assessment; pre-clinical and clinical research; regulatory authorization and medicines access; and engagement with patients, policy makers, and the public. The InterAcademy Partnership (IAP) identified options for action for sharing good practice and building collaboration within the scientific community and with other stakeholders worldwide.

Transient reprogramming of postnatal cardiomyocytes to a dedifferentiated state.

In contrast to mammals, lower vertebrates are capable of extraordinary myocardial regeneration thanks to the ability of their cardiomyocytes to undergo transient dedifferentiation and proliferation. Somatic cells can be temporarily reprogrammed to a proliferative, dedifferentiated state through forced expression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM). Here, we aimed to induce transient reprogramming of mammalian cardiomyocytes in vitro utilising an OSKM-encoding non-integrating vector. Reprogramming factor expression in postnatal rat and mouse cardiomyocytes triggered rapid but limited cell dedifferentiation. Concomitantly, a significant increase in cell viability, cell cycle related gene expression and Ki67 positive cells was observed consistent with an enhanced cell cycle activation. The transient nature of this partial reprogramming was confirmed as cardiomyocyte-specific cell morphology, gene expression and contractile activity were spontaneously recovered by day 15 after viral transduction. This study provides the first evidence that adenoviral OSKM delivery can induce partial reprogramming of postnatal cardiomyocytes. Therefore, adenoviral mediated transient reprogramming could be a novel and feasible strategy to recapitulate the regenerative mechanisms of lower vertebrates.

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

Detecting WDM visible light signals by a single multi-color photodiode with MIMO processing.

For the first time, to the best of our knowledge, we experimentally demonstrate that multiple-input-multiple-output (MIMO) processing allows using a single photodiode to detect simultaneously a wavelength-division multiplexing (WDM) visible light communications (VLC) signal. The photodiode has a triple junction, and when it is illuminated by a WDM signal, the junctions produce inherently three photocurrents that are unusable for detecting any of the WDM signals. However, by means of linear MIMO processing, we are able to recover the transmitted signals exactly. Bit error rate measurements confirm the effectiveness of the proposed solution. This opens a new scenario for practical WDM-VLC systems.

TNAP limits TGF-ß-dependent cardiac and skeletal muscle fibrosis by inactivating the SMAD2/3 transcription factors.

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor ß (TGF-ß) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-ß signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-ß signalling and likely represents a mechanism to limit fibrosis.

Correction: Magic-Factor 1, a Partial Agonist of Met, Induces Muscle Hypertrophy by Protecting Myogenic Progenitors from Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0003223.].

Advances in stem cell research and therapeutic development.

Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.

Combined Notch and PDGF Signaling Enhances Migration and Expression of Stem Cell Markers while Inducing Perivascular Cell Features in Muscle Satellite Cells.

Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy.

Non-viral, Tumor-free Induction of Transient Cell Reprogramming in Mouse Skeletal Muscle to Enhance Tissue Regeneration.

Overexpression of Oct3/4, Klf4, Sox2, and c-Myc (OKSM) transcription factors can de-differentiate adult cells in vivo. While sustained OKSM expression triggers tumorigenesis through uncontrolled proliferation of toti- and pluripotent cells, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratomas. We sought to transiently reprogram cells within mouse skeletal muscle with a localized injection of plasmid DNA encoding OKSM (pOKSM), and we hypothesized that the generation of proliferative intermediates would enhance tissue regeneration after injury. Intramuscular pOKSM administration rapidly upregulated pluripotency (Nanog, Ecat1, and Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various strains. Mononucleated cells expressing such markers appeared in clusters among myofibers, proliferated only transiently, and did not lead to dysplasia or tumorigenesis for at least 120 days. Nanog was also upregulated in the gastrocnemius when pOKSM was administered 7 days after surgically sectioning its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centronucleated myofibers and reduced fibrosis. These results suggest that transient in vivo reprogramming could develop into a novel strategy toward the acceleration of tissue regeneration after injury, based on the induction of transiently proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted.