Publications by authors named "Maurilio Sampaolesi"

124 Publications

Therapeutic Implications of miRNAs for Muscle-Wasting Conditions.

Cells 2021 11 5;10(11). Epub 2021 Nov 5.

Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that are mainly involved in translational repression by binding to specific messenger RNAs. Recently, miRNAs have emerged as biomarkers, relevant for a multitude of pathophysiological conditions, and cells can selectively sort miRNAs into extracellular vesicles for paracrine and endocrine effects. In the overall context of muscle-wasting conditions, a multitude of miRNAs has been implied as being responsible for the typical dysregulation of anabolic and catabolic pathways. In general, chronic muscle disorders are associated with the main characteristic of a substantial loss in muscle mass. Muscular dystrophies (MDs) are a group of genetic diseases that cause muscle weakness and degeneration. Typically, MDs are caused by mutations in those genes responsible for upholding the integrity of muscle structure and function. Recently, the dysregulation of miRNA levels in such pathological conditions has been reported. This revelation is imperative for both MDs and other muscle-wasting conditions, such as sarcopenia and cancer cachexia. The expression levels of miRNAs have immense potential for use as potential diagnostic, prognostic and therapeutic biomarkers. Understanding the role of miRNAs in muscle-wasting conditions may lead to the development of novel strategies for the improvement of patient management.
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http://dx.doi.org/10.3390/cells10113035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616481PMC
November 2021

Incomplete Assembly of the Dystrophin-Associated Protein Complex in 2D and 3D-Cultured Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Front Cell Dev Biol 2021 4;9:737840. Epub 2021 Nov 4.

Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are increasingly used to study genetic diseases on a human background. However, the lack of a fully mature adult cardiomyocyte phenotype of hiPSC-CM may be limiting the scope of these studies. Muscular dystrophies and concomitant cardiomyopathies result from mutations in genes encoding proteins of the dystrophin-associated protein complex (DAPC), which is a multi-protein membrane-spanning complex. We examined the expression of DAPC components in hiPSC-CM, which underwent maturation in 2D and 3D culture protocols. The results were compared with human adult cardiac tissue and isolated cardiomyocytes. We found that similarly to adult cardiomyocytes, hiPSC-CM express dystrophin, in line with previous studies on Duchenne's disease. β-dystroglycan was also expressed, but, contrary to findings in adult cardiomyocytes, none of the sarcoglycans nor α-dystroglycan were, despite the presence of their mRNA. In conclusion, despite the robust expression of dystrophin, the absence of several other DAPC protein components cautions for reliance on commonly used protocols for hiPSC-CM maturation for functional assessment of the complete DAPC.
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http://dx.doi.org/10.3389/fcell.2021.737840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599983PMC
November 2021

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.

J Vis Exp 2021 09 7(175). Epub 2021 Sep 7.

Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute, KU Leuven - University of Leuven; VIB Center for Brain & Disease Research, Laboratory of Neurobiology, ;

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.
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http://dx.doi.org/10.3791/62959DOI Listing
September 2021

Improved functionality and potency of next generation BinMLV viral vectors toward safer gene therapy.

Mol Ther Methods Clin Dev 2021 Dec 21;23:51-67. Epub 2021 Jul 21.

Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmacological and Pharmaceutical Sciences, KU Leuven, 3000 Leuven, Belgium.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLV was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLV vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLV-EFS produced like MLV and efficiently transduced laboratory cells and primary human CD34 hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLV transduction, while this remained unaffected when applying MLV. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLV-EFS. Taken together, our results underscore the potential of MLV-EFS as a clinically relevant viral vector for gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.
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http://dx.doi.org/10.1016/j.omtm.2021.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433069PMC
December 2021

Upregulation of miR181a/miR212 Improves Myogenic Commitment in Murine Fusion-Negative Rhabdomyosarcoma.

Front Physiol 2021 6;12:701354. Epub 2021 Aug 6.

Stem Cell Institute Leuven, KU Leuven, Leuven, Belgium.

Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications.
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http://dx.doi.org/10.3389/fphys.2021.701354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378536PMC
August 2021

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

Front Genet 2021 2;12:702547. Epub 2021 Aug 2.

Department of Toxicogenomics, Maastricht University Medical Centre, Maastricht, Netherlands.

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 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.
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http://dx.doi.org/10.3389/fgene.2021.702547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365145PMC
August 2021

Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a NOTCH-dependent manner.

Cell Death Dis 2021 07 5;12(7):677. Epub 2021 Jul 5.

Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, 3000, Leuven, Belgium.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.
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http://dx.doi.org/10.1038/s41419-021-03936-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257578PMC
July 2021

Actuation enhances patterning in human neural tube organoids.

Nat Commun 2021 05 27;12(1):3192. Epub 2021 May 27.

Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.
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http://dx.doi.org/10.1038/s41467-021-22952-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159931PMC
May 2021

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Stem Cell Reports 2021 09 22;16(9):2213-2227. Epub 2021 Apr 22.

KU Leuven - University of Leuven, Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute, Leuven, Belgium; VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium. Electronic address:

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.
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http://dx.doi.org/10.1016/j.stemcr.2021.03.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452598PMC
September 2021

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization.

Int J Mol Sci 2021 Feb 16;22(4). Epub 2021 Feb 16.

Human Anatomy Unit, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, 27100 Pavia, Italy.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.
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http://dx.doi.org/10.3390/ijms22041939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920023PMC
February 2021

Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation.

Cell Stem Cell 2021 06 19;28(6):1125-1135.e7. Epub 2021 Feb 19.

Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France. Electronic address:

Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations.
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http://dx.doi.org/10.1016/j.stem.2021.01.017DOI Listing
June 2021

Isolation of Mammalian Mesoangioblasts: A Subset of Pericytes with Myogenic Potential.

Methods Mol Biol 2021 ;2235:155-167

Translational Cardiomyology Laboratory, Stem Cell Institute, Department of Development and Regeneration, KU Leuven, Leuven, Belgium.

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.
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http://dx.doi.org/10.1007/978-1-0716-1056-5_11DOI Listing
March 2021

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification.

Cell Death Differ 2021 04 28;28(4):1193-1207. Epub 2020 Oct 28.

International Clinical Research Center (ICRC) of St Anne's University Hospital, CZ-65691, Brno, Czech Republic.

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.
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http://dx.doi.org/10.1038/s41418-020-00643-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027678PMC
April 2021

Muscle Microbiopsy to Delineate Stem Cell Involvement in Young Patients: A Novel Approach for Children With Cerebral Palsy.

Front Physiol 2020 6;11:945. Epub 2020 Aug 6.

Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium.

Cerebral palsy (CP), the single largest cause of childhood physical disability, is characterized firstly by a lesion in the immature brain, and secondly by musculoskeletal problems that progress with age. Previous research reported altered muscle properties, such as reduced volume and satellite cell (SC) numbers and hypertrophic extracellular matrix compared to typically developing (TD) children (>10 years). Unfortunately, data on younger CP patients are scarce and studies on SCs and other muscle stem cells in CP are insufficient or lacking. Therefore, it remains difficult to understand the early onset and trajectory of altered muscle properties in growing CP children. Because muscle stem cells are responsible for postnatal growth, repair and remodeling, multiple adult stem cell populations from young CP children could play a role in altered muscle development. To this end, new methods for studying muscle samples of young children, valid to delineate the features and to elucidate the regenerative potential of muscle tissue, are necessary. Using minimal invasive muscle microbiopsy, which was applied in young subjects under general anaesthesia for the first time, we aimed to isolate and characterize muscle stem cell-derived progenitors of TD children and patients with CP. Data of 15 CP patients, 3-9 years old, and 5 aged-matched TD children were reported. The muscle microbiopsy technique was tolerated well in all participants. Through the explant technique, we provided muscle stem cell-derived progenitors from the Via fluorescent activated cell sorting, using surface markers CD56, ALP, and PDGFRa, we obtained SC-derived progenitors, mesoangioblasts and fibro-adipogenic progenitors, respectively. Adipogenic, skeletal, and smooth muscle differentiation assays confirmed the cell identity and ability to give rise to different cell types after appropriate stimuli. Myogenic differentiation in CP SC-derived progenitors showed enhanced fusion index and altered myotube formation based on MYOSIN HEAVY CHAIN expression, as well as disorganization of nuclear spreading, which were not observed in TD myotubes. In conclusion, the microbiopsy technique allows more focused muscle research in young CP patients. Current results show altered differentiation abilities of muscle stem cell-derived progenitors and support the hypothesis of their involvement in CP-altered muscle growth.
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http://dx.doi.org/10.3389/fphys.2020.00945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424076PMC
August 2020

MICAL2 is essential for myogenic lineage commitment.

Cell Death Dis 2020 08 18;11(8):654. Epub 2020 Aug 18.

Translational Cardiomyology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.
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http://dx.doi.org/10.1038/s41419-020-02886-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434877PMC
August 2020

Both ghrelin deletion and unacylated ghrelin overexpression preserve muscles in aging mice.

Aging (Albany NY) 2020 07 26;12(14):13939-13957. Epub 2020 Jul 26.

Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy.

Sarcopenia, the decline in muscle mass and functionality during aging, might arise from age-associated endocrine dysfunction. Ghrelin is a hormone circulating in both acylated (AG) and unacylated (UnAG) forms with anti-atrophic activity on skeletal muscle. Here, we show that not only lifelong overexpression of UnAG (Tg) in mice, but also the deletion of ghrelin gene ( KO) attenuated the age-associated muscle atrophy and functionality decline, as well as systemic inflammation. Yet, the aging of Tg and KO mice occurs with different dynamics: while old Tg mice seem to preserve the characteristics of young animals, KO mice features deteriorate with aging. However, young KO mice show more favorable traits compared to WT animals that result, on the whole, in better performances in aged KO animals. Treatment with pharmacological doses of UnAG improved muscle performance in old mice without modifying the feeding behavior, body weight, and adipose tissue mass. The antiatrophic effect on muscle mass did not correlate with modifications of protein catabolism. However, UnAG treatment induced a strong shift towards oxidative metabolism in muscle. Altogether, these data confirmed and expanded some of the previously reported findings and advocate for the design of UnAG analogs to treat sarcopenia.
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http://dx.doi.org/10.18632/aging.103802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425472PMC
July 2020

In vivo stem cell tracking using scintigraphy in a canine model of DMD.

Sci Rep 2020 06 30;10(1):10681. Epub 2020 Jun 30.

Inserm U955-E10, IMRB, Université Paris Est Créteil. Ecole nationale vétérinaire d'Alfort, 94700, Maisons-Alfort, France.

One of the main challenges in cell therapy for muscle diseases is to efficiently target the muscle. To address this issue and achieve better understanding of in vivo cell fate, we evaluated the relevance of a non-invasive cell tracking method in the Golden Retriever Muscular Dystrophy (GRMD) model, a well-recognised model of Duchenne Muscular Dystrophy (DMD). Mesoangioblasts were directly labelled with In-oxine, and injected through one of the femoral arteries. The scintigraphy images obtained provided the first quantitative mapping of the immediate biodistribution of mesoangioblasts in a large animal model of DMD. The results revealed that cells were trapped by the first capillary filters: the injected limb and the lung. During the days following injection, radioactivity was redistributed to the liver. In vitro studies, performed with the same cells prepared for injecting the animal, revealed prominent cell death and In release. In vivo, cell death resulted in In release into the vasculature that was taken up by the liver, resulting in a non-specific and non-cell-bound radioactive signal. Indirect labelling methods would be an attractive alternative to track cells on the mid- and long-term.
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http://dx.doi.org/10.1038/s41598-020-66388-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327062PMC
June 2020

Myoblasts Tracking Using the Sodium Iodide Symporter Gene Expression in Dogs.

Mol Ther Methods Clin Dev 2020 Jun 9;17:317-327. Epub 2020 Jan 9.

INSERM U955-E10, IMRB, Université Paris Est Créteil, Ecole nationale vétérinaire d'Alfort, 94700 Maisons-Alfort, France.

Stem cell-based therapies are a promising approach for the treatment of degenerative muscular diseases; however, clinical trials have shown inconclusive and even disappointing results so far. Noninvasive cell monitoring by medicine imaging could improve the understanding of the survival and biodistribution of cells following injection. In this study, we assessed the canine sodium iodide symporter (cNIS) reporter gene as an imaging tool to track by single-photon emission computed tomography (SPECT/CT) transduced canine myoblasts after intramuscular (IM) administrations in dogs. cNIS-expressing cells kept their myogenic capacities and showed strong Tc-pertechnetate (TcO ) uptake efficiency both and . cNIS expression allowed visualization of cells by SPECT/CT along time: 4 h, 48 h, 7 days, and 30 days after IM injection; biopsies collected 30 days post administration showed myofiber's membranes expressing cNIS. This study demonstrates that NIS can be used as a reporter to track cells in the skeletal muscle of large animals. Our results set a proof of concept of the benefits NIS-tracking tool may bring to the already challenging cell-based therapies arena in myopathies and pave the way to a more efficient translation to the clinical setting from more accurate pre-clinical results.
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http://dx.doi.org/10.1016/j.omtm.2019.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293195PMC
June 2020

Frizzled related protein deficiency impairs muscle strength, gait and calpain 3 levels.

Orphanet J Rare Dis 2020 05 24;15(1):119. Epub 2020 May 24.

Biodonostia Health Research Institute, Neurosciences Area, San Sebastian, Spain.

Background: Limb-girdle muscular dystrophy recessive 1 calpain3-related (LGMDR1), previously known as LGMD2A, is a disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness and muscle degeneration. Frizzled related protein (FRZB), upregulated in LGMDR1, was identified as a key regulator of the crosstalk between Wnt and integrin signalling pathways. FRZB gene silencing showed a recovery in the expression of some of the costamere protein levels in myotubes.

Results: Here, we performed a comprehensive characterization of Frzb mice muscles to study the absence of Frzb in skeletal muscle and eventual links with the molecular characteristics of LGMDR1 patient muscles. Frzb mice showed reduced muscle size and strength. Gait analysis showed that Frzb mice moved more slowly but no impaired regeneration capacity was observed after muscle injury. Additionally, Frzb mice muscle showed an increased number of mesoangioblasts. Lack of Frzb gene in Frzb mice and its increased expression in LGMDR1 patients, showed contrary regulation of Rora, Slc16a1, Tfrc and Capn3 genes. The reciprocal regulation of Frzb and Capn3 genes further supports this axis as a potential target for LGMDR1 patients.

Conclusions: Our data confirm a role for Frzb in the regulation of Rora, Slc16a1, Tfrc, and Capn3 genes in muscle cells. In vivo, reduced muscle strength and gait in the Frzb mice are intriguing features. The reciprocal relationship between FRZB and CAPN3 further supports a key role for this axis in patients with LGMDR1.
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http://dx.doi.org/10.1186/s13023-020-01372-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7245871PMC
May 2020

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Cell Rep 2020 05;31(5):107597

Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, 3000 Leuven, Belgium; Human Anatomy Unit, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, 27100 Pavia, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2020.107597DOI Listing
May 2020

Comprehensive Overview of Non-coding RNAs in Cardiac Development.

Adv Exp Med Biol 2020 ;1229:197-211

Translational Cardiomyology Laboratory, Stem Cell Research Institute, Stem Cell Biology and Embryology Unit, Department of Development and Regeneration, KU Leuven, Leuven, Belgium.

Cardiac development in the human embryo is characterized by the interactions of several transcription and growth factors leading the heart from a primordial linear tube into a synchronous contractile four-chamber organ. Studies on cardiogenesis showed that cell proliferation, differentiation, fate specification and morphogenesis are spatiotemporally coordinated by cell-cell interactions and intracellular signalling cross-talks. In recent years, research has focused on a class of inter- and intra-cellular modulators called non-coding RNAs (ncRNAs), transcribed from the noncoding portion of the DNA and involved in the proper formation of the heart. In this chapter, we will summarize the current state of the art on the roles of three major forms of ncRNAs [microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs)] in orchestrating the four sequential phases of cardiac organogenesis.
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http://dx.doi.org/10.1007/978-981-15-1671-9_11DOI Listing
July 2020

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

Int J Mol Sci 2020 Apr 5;21(7). Epub 2020 Apr 5.

Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium.

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.
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http://dx.doi.org/10.3390/ijms21072525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177401PMC
April 2020

Interleukin-4 administration improves muscle function, adult myogenesis, and lifespan of colon carcinoma-bearing mice.

J Cachexia Sarcopenia Muscle 2020 06 27;11(3):783-801. Epub 2020 Feb 27.

Translational Cardiomyology, Stem Cell Biology and Embryology, Department of Development and Regeneration, University Hospital Gasthuisberg, Leuven, Belgium.

Background: Anorexia, body wasting, inflammation, muscle, and adipose tissue loss are hallmarks of cancer cachexia, a syndrome that affects the majority of cancer patients, impairing their ability to endure chemotherapeutic therapies and reducing their lifespan. In the last 10 years, alterations of protein turnover and impairment of adult myogenesis have been proposed as major contributing factors.

Methods: Muscle stem cells, including satellite cells, mesoangioblasts, and fibroadipogenic progenitors, were isolated and characterized from C26 colon carcinoma-bearing (C26) mice. Circulating levels of interleukin-4/13 (IL4/IL13) were analysed by ELISA, and the effects of IL4 on muscle mass and function, protein synthesis, muscle regeneration, and myogenic progenitor cell number were analysed at both functional (treadmill and grip test) and molecular levels (qRT-PCR, immunofluorescence analysis, surface sensing of translation, and western blot). The Kaplan-Meier test was used to analyse the survival curve of IL4-treated and IL4-untreated C26 mice.

Results: The administration of IL4 to C26 mice rescued muscle mass by increasing protein synthesis. The IL4 treatment improved performances and prolonged survival of C26 mice. IL4 administration re-established both number and function of satellite cells and fibroadipogenic progenitors without affecting mesoangioblasts in C26 mice, rescuing myogenesis. Upon IL4 treatment, a high number of cytotoxic lymphocytes and type II macrophages were observed with a subsequent increase in necrotic areas of C26 tumours.

Conclusions: The results here presented shed new light on IL4 signalling during muscle wasting and early stages of muscle regeneration that explain the beneficial effect observed in IL4-treated C26 mice. These findings might aid to develop therapeutic approaches to improve mobility and quality of life in cachectic patients.
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http://dx.doi.org/10.1002/jcsm.12539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296260PMC
June 2020

Activator Protein-1 Transcriptional Activity Drives Soluble Micrograft-Mediated Cell Migration and Promotes the Matrix Remodeling Machinery.

Stem Cells Int 2019 31;2019:6461580. Epub 2019 Dec 31.

Department of Development and Regeneration, Stem Cell Institute, KU Leuven, B-3000 Leuven, Belgium.

Impaired wound healing and tissue regeneration have severe consequences on the patient's quality of life. Micrograft therapies are emerging as promising and affordable alternatives to improve skin regeneration by enhancing the endogenous wound repair processes. However, the molecular mechanisms underpinning the beneficial effects of the micrograft treatments remain largely unknown. In this study, we identified the active protein-1 (AP-1) member Fos-related antigen-1 (Fra-1) to play a central role in the extracellular signal-regulated kinase- (ERK-) mediated enhanced cell migratory capacity of soluble micrograft-treated mouse adult fibroblasts and in the human keratinocyte cell model. Accordingly, we show that increased micrograft-dependent cell migration and matrix metalloprotease activity is abolished upon inhibition of AP-1. Furthermore, soluble micrograft treatment leads to increased expression and posttranslational phosphorylation of Fra-1 and c-Jun, resulting in the upregulation of wound healing-associated genes mainly involved in the regulation of cell migration. Collectively, our work provides insights into the molecular mechanisms behind the cell-free micrograft treatment, which might contribute to future advances in wound repair therapies.
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http://dx.doi.org/10.1155/2019/6461580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012246PMC
December 2019

Human fetal mesoangioblasts reveal tissue-dependent transcriptional signatures.

Stem Cells Transl Med 2020 05 23;9(5):575-589. Epub 2020 Jan 23.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Mesoangioblasts (MABs) derived from adult skeletal muscles are well-studied adult stem/progenitor cells that already entered clinical trials for muscle regeneration in genetic diseases; however, the transcriptional identity of human fetal MABs (fMABs) remains largely unknown. Herein we analyzed the transcriptome of MABs isolated according to canonical markers from fetal atrium, ventricle, aorta, and skeletal muscles (from 9.5 to 13 weeks of age) to uncover specific gene signatures correlating with their peculiar myogenic differentiation properties inherent to their tissue of origin. RNA-seq analysis revealed for the first time that human MABs from fetal aorta, cardiac (atrial and ventricular), and skeletal muscles display subsets of differentially expressed genes likely representing distinct expression signatures indicative of their original tissue. Identified GO biological processes and KEGG pathways likely account for their distinct differentiation outcomes and provide a set of critical genes possibly predicting future specific differentiation outcomes. This study reveals novel information regarding the potential of human fMABs that may help to improve specific differentiation outcomes relevant for therapeutic muscle regeneration.
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http://dx.doi.org/10.1002/sctm.19-0209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180296PMC
May 2020

Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy.

Stem Cell Res Ther 2019 12 21;10(1):405. Epub 2019 Dec 21.

School for Developmental Biology and Oncology (GROW), Maastricht University Medical Centre+, P.O. box 616, 6200MD, Maastricht, The Netherlands.

Background: Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy.

Methods: We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity.

Results: This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity.

Conclusions: Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.
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http://dx.doi.org/10.1186/s13287-019-1510-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925445PMC
December 2019

Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration.

Cell Death Differ 2020 05 25;27(5):1520-1538. Epub 2019 Oct 25.

Department of Development and Regeneration, Stem Cell Institute, KU Leuven, B-3000, Leuven, Belgium.

Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.
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http://dx.doi.org/10.1038/s41418-019-0433-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206041PMC
May 2020

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes.

Transl Psychiatry 2019 08 21;9(1):200. Epub 2019 Aug 21.

Stem Cell Institute Leuven, Dept. of Development and Regeneration, KU Leuven, Leuven, Belgium.

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.
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http://dx.doi.org/10.1038/s41398-019-0535-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704264PMC
August 2019

(Epi)genetic Modifications in Myogenic Stem Cells: From Novel Insights to Therapeutic Perspectives.

Cells 2019 05 9;8(5). Epub 2019 May 9.

Translational Cardiomyology Lab, Department of Development and Regeneration, Stem Cell Institute Leuven, 3000 KU Leuven, Belgium.

The skeletal muscle is considered to be an ideal target for stem cell therapy as it has an inherent regenerative capacity. Upon injury, the satellite cells, muscle stem cells that reside under the basal lamina of the myofibres, start to differentiate in order to reconstitute the myofibres while maintaining the initial stem cell pool. In recent years, it has become more and more evident that epigenetic mechanisms such as histon modifications, DNA methylations and microRNA modulations play a pivatol role in this differentiation process. By understanding the mechanisms behind myogenesis, researchers are able to use this knowledge to enhance the differentiation and engraftment potential of different muscle stem cells. Besides manipulation on an epigenetic level, recent advances in the field of genome-engineering allow site-specific modifications in the genome of these stem cells. Combining epigenetic control of the stem cell fate with the ability to site-specifically correct mutations or add genes for further cell control, can increase the use of stem cells as treatment of muscular dystrophies drastically. In this review, we will discuss the advances that have been made in genome-engineering and the epigenetic regulation of muscle stem cells and how this knowledge can help to get stem cell therapy to its full potential.
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http://dx.doi.org/10.3390/cells8050429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562881PMC
May 2019
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