Cardiomyocyte differentiation from human induced pluripotent stem cells is delayed following knockout of Bcl-2.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival and autophagy. Less known is its involvement in the differentiation of cardiomyocytes. As a consequence, mechanisms by which Bcl-2 contributes to cardiac differentiation remain to be elucidated. To address this, we used CRISPR/Cas9 to knockout (KO) BCL2 in human induced pluripotent stem cells (hiPSCs) and investigated the consequence of this KO for differentiation towards cardiomyocytes. Our results indicate that differentiation of hiPSCs to cardiomyocytes was delayed following BCL2 KO. This was not related to the canonical anti-apoptotic function of Bcl-2. This delay led to reduced expression and activity of the cardiomyocyte Ca2+ toolkit. Finally, Bcl-2 KO reduced c-Myc expression and nuclear localization in the early phase of the cardiac differentiation process, which accounts at least in part for the observed delay in the cardiac differentiation. These results suggest that there is a central role for Bcl-2 in cardiomyocyte differentiation and maturation.

Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity.

Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

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

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.

Comprehensive Overview of Non-coding RNAs in Cardiac Development.

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.

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

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.

Growth Factor Screening in Dystrophic Muscles Reveals PDGFB/PDGFRB-Mediated Migration of Interstitial Stem Cells.

Progressive muscle degeneration followed by dilated cardiomyopathy is a hallmark of muscular dystrophy. Stem cell therapy is suggested to replace diseased myofibers by healthy myofibers, although so far, we are faced by low efficiencies of migration and engraftment of stem cells. Chemokines are signalling proteins guiding cell migration and have been shown to tightly regulate muscle tissue repair. We sought to determine which chemokines are expressed in dystrophic muscles undergoing tissue remodelling. Therefore, we analysed the expression of chemokines and chemokine receptors in skeletal and cardiac muscles from Sarcoglycan-α null, Sarcoglycan-ß null and immunodeficient Sgcß-null mice. We found that several chemokines are dysregulated in dystrophic muscles. We further show that one of these, platelet-derived growth factor-B, promotes interstitial stem cell migration. This finding provides perspective to an approachable mechanism for improving stem cell homing towards dystrophic muscles.

Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb-girdle muscular dystrophy type 2E.

INTRODUCTION: Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the ß-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. ß-Sarcoglycan-deficient (Sgcb-null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. METHODS: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1-month-old (clinical onset of the disease) and 7-month-old control mice (C57Bl/6J, Rag2/γc-null) and immunocompetent and immunodeficient dystrophic mice (Sgcb-null and Sgcb/Rag2/γc-null, respectively). RESULTS: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc-null muscles showed a significant reduction of alkaline phosphate-positive mesoangioblasts. DISCUSSION: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve, 2018.

Interactions between microRNAs and long non-coding RNAs in cardiac development and repair.

Non-coding RNAs (ncRNAs) are emerging players in muscle regulation. Based on their length and differences in molecular structure, ncRNAs are subdivided into several categories including small interfering RNAs, stable non-coding RNAs, microRNAs (miRs), long non-coding RNAs (lncRNAs), and circular RNAs. miRs and lncRNAs are able to post-transcriptionally regulate many genes and bring into play several traits simultaneously due to a myriad of different targets. Recent studies have emphasized their importance in cardiac regeneration and repair. As their altered expression affects cardiac function, miRs and lncRNAs could be potential targets for therapeutic intervention. In this context, miR- and lncRNA-based gene therapies are an interesting field for harnessing the complexity of ncRNA-based therapeutic approaches in cardiac diseases. In this review we will focus on lncRNA- and miR-driven regulations of cardiac development and repair. Finally, we will summarize miRs and lncRNAs as promising candidates for the treatment of heart diseases.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

Ether-Oxygen Containing Electrospun Microfibrous and Sub-Microfibrous Scaffolds Based on Poly(butylene 1,4-cyclohexanedicarboxylate) for Skeletal Muscle Tissue Engineering.

We report the study of novel biodegradable electrospun scaffolds from poly(butylene 1,4-cyclohexandicarboxylate-co-triethylene cyclohexanedicarboxylate) (P(BCE-co-TECE)) as support for in vitro and in vivo muscle tissue regeneration. We demonstrate that chemical composition, i.e., the amount of TECE co-units (constituted of polyethylene glycol-like moieties), and fibre morphology, i.e., aligned microfibrous or sub-microfibrous scaffolds, are crucial in determining the material biocompatibility. Indeed, the presence of ether linkages influences surface wettability, mechanical properties, hydrolytic degradation rate, and density of cell anchoring points of the studied materials. On the other hand, electrospun scaffolds improve cell adhesion, proliferation, and differentiation by favouring cell alignment along fibre direction (fibre morphology), also allowing for better cell infiltration and oxygen and nutrient diffusion (fibre size). Overall, C2C12 myogenic cells highly differentiated into mature myotubes when cultured on microfibres realised with the copolymer richest in TECE co-units (micro-P73 mat). Lastly, when transplanted in the tibialis anterior muscles of healthy, injured, or dystrophic mice, micro-P73 mat appeared highly vascularised, colonised by murine cells and perfectly integrated with host muscles, thus confirming the suitability of P(BCE-co-TECE) scaffolds as substrates for skeletal muscle tissue engineering.

Myomir dysregulation and reactive oxygen species in aged human satellite cells.

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

Myogenic induction of adult and pluripotent stem cells using recombinant proteins.

Met Activating Genetically Improved Chimeric Factor 1 (Magic-F1) is a human recombinant protein, derived from dimerization of the receptor-binding domain of hepatocyte growth factor. Previous experiments demonstrate that in transgenic mice, the skeletal muscle specific expression of Magic-F1 can induce a constitutive muscular hypertrophy, improving running performance and accelerating muscle regeneration after injury. In order to evaluate the therapeutic potential of Magic-F1, we tested its effect on multipotent and pluripotent stem cells. In murine mesoangioblasts (adult vessel-associated stem cells), the presence of Magic-F1 did not alter their osteogenic, adipogenic or smooth muscle differentiation ability. However, when analyzing their myogenic potential, mesoangioblasts expressing Magic-F1 differentiated spontaneously into myotubes. Finally, Magic-F1 inducible cassette was inserted into a murine embryonic stem cell line by homologous recombination. When embryonic stem cells were subjected to myogenic differentiation, the presence of Magic-F1 resulted in the upregulation of Pax3 and Pax7 that enhanced the myogenic commitment of transgenic pluripotent stem cells. Taken together our results candidate Magic-F1 as a potent myogenic stimulator, able to enhance muscular differentiation from both adult and pluripotent stem cells.

Fate choice of post-natal mesoderm progenitors: skeletal versus cardiac muscle plasticity.

Regenerative medicine for skeletal and cardiac muscles still constitutes a fascinating and ambitious frontier. In this perspective, understanding the possibilities of intrinsic cell plasticity, present in post-natal muscles, is vital to define and improve novel therapeutic strategies for acute and chronic diseases. In addition, many somatic stem cells are now crossing the boundaries of basic/translational research to enter the first clinical trials. However, it is still an open question whether a lineage switch between skeletal and cardiac adult myogenesis is possible. Therefore, this review focuses on resident somatic stem cells of post-natal skeletal and cardiac muscles and their plastic potential toward the two lineages. Furthermore, examples of myogenic lineage switch in adult stem cells are also reported and discussed.

Role of Inflammation in Muscle Homeostasis and Myogenesis.

Skeletal muscle mass is subject to rapid changes according to growth stimuli inducing both hypertrophy, through increased protein synthesis, and hyperplasia, activating the myogenic program. Muscle wasting, characteristic of several pathological states associated with local or systemic inflammation, has been for long considered to rely on the alteration of myofiber intracellular pathways regulated by both hormones and cytokines, eventually leading to impaired anabolism and increased protein breakdown. However, there are increasing evidences that even alterations of the myogenic/regenerative program play a role in the onset of muscle wasting, even though the precise mechanisms involved are far from being fully elucidated. The comprehension of the links potentially occurring between impaired myogenesis and increased catabolism would allow the definition of effective strategies aimed at counteracting muscle wasting. The first part of this review gives an overview of skeletal muscle intracellular pathways determining fiber size, while the second part considers the cells and the regulatory pathways involved in the myogenic program. In both parts are discussed the evidences supporting the role of inflammation in impairing muscle homeostasis and myogenesis, potentially determining muscle atrophy.

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo.

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.

Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.

Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Altered functional differentiation of mesoangioblasts in a genetic myopathy.

Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from ß-sarcoglycan-null mice (ßSG(-/-)), a murine model of genetic myopathy with early myocardial involvement. Although complementation with ßSG gene was inconsequential, knock-in of miRNA669a (missing in ßSG(-/-) cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of ßSG(-/-) cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca(2+)-handling properties of ßSG(-/-) cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca(2+)-spark properties and RyR isoform expression, distinguished ßSG(-/-) cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in ßSG(-/-) cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by ßSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.