Biology subjects

Cossu, G.

Publications and source records attributed to Cossu, G..

7 recordsLinked to original sources

bioRxiv2026

Systemic Delivery of Human Mesangioblasts mediated by a Nanofiber Scaffold restores Dystrophin Expression in Immunodeficient Dystrophic Mice.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@66d0eaorg.highwire.dtl.DTLVardef@1dea3forg.highwire.dtl.DTLVardef@1fdf837org.highwire.dtl.DTLVardef@b2f86a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG

bioengineering↗

bioRxiv2025

Mini-agrin prevents calcium leakage and restores the dystrophin complex.

Muscle cell death in muscular dystrophies depends upon calcium ion (Ca++) leakage through sarcolemma and sarcoplasmic reticulum, triggered by muscle stretch during eccentric contraction. We show here that Ca++ spikes are detected in dystrophic myogenic cells in culture since early differentiation, before sarcomere assembly and contraction. Healthy and genetically corrected dystrophic myotubes do not display Ca++ spikes which are blocked by co-culturing DMD myogenic cells with embryonic mouse motoneurons or treating them with agrin proteoglycan. Same effect is elicited by a muscle spliced, COOH peptide of agrin (termed here mini-agrin) that interacts with dystroglycan, favouring its binding to the basal lamina. Lack of dystrophin in DMD myotubes results in decreased expression of CaV1.1 (CACNA1S), a Ca++ sensor component of the Dihydropyridine Receptor (DHPR) complex, known to regulate Ryanodine Receptor 1 (RyR1). These events explain the emergence of Ca++ spikes. Mini-agrin addition to medium, or lentivector-mediated mini-agrin expression in transplanted cells in vivo, stabilize the expression of CaV1.1 on the membrane. This leads to disappearance of Ca++ spikes and to reappearance of -dystroglycan, -sarcoglycan and n-NOS, indicating the reconstitution of the dystrophin complex in the absence of dystrophin. These findings unveil a novel regulatory mechanism and offer a new therapeutic opportunity for targeting calcium ion influx as a co-treatment strategy.

cell biology↗

bioRxiv2025

Slit1 -a MET target gene in the embryonic limbs, prevents premature differentiation during mammalian myogenesis.

Skeletal myogenesis requires precise spatio-temporal regulation of molecular signals during prenatal and postnatal development. Prenatal myogenesis in mouse commences at embryonic day (E) 9.5, characterized by the expression of PAX3 -a key myogenic regulator, and its target MET, in the embryonic muscle progenitor cells (EMPCs). These EMPCs delaminate from dermomyotome in the somites and migrate to designated areas such as the developing embryonic limbs and diaphragm. The trajectory of their migration is directed and limited by spatio-temporal availability of the MET ligand (HGF). Given its periodic expression during embryonic myogenesis and its recent association with familial arthrogryposis, it is important to identify additional non-migratory functions of MET signaling. We find conditional loss of Met in the Pax3/somitic lineage affects survival in early neonatal stages because amuscular diaphragms cause respiratory distress. Impaired progenitor migration in conditional Met knockouts (cMetKO) results in highly dysplastic muscles in neonates compared to muscleless limbs observed in previous mutants. Additionally, using cMetKO embryos (E11.5) we identify Slit1 as a novel target of MET that is downregulated in the mutant limb buds. Pharmacological modulation with SU11274, in vitro, confirms Slit1 as a MET responsive gene, which if knocked down in myoblasts leads to precocious myogenic differentiation. Similarly, cMetKO embryos, having reduced Slit1 expression, show greater myotomal differentiation and compact organization, compared to wildtype embryos. While Slit1 emerges as a MET target that represses precocious switch to myogenic differentiation, molecular intricacies of MET-mediated regulation of Slit1 and their spatio-temporal dynamic in fine-tuning myogenesis in the nascent limbs needs further examination.

developmental biology↗

bioRxiv2019

Transient Reprogramming of Neonatal Cardiomyocytes to a Proliferative Dedifferentiated State

Zebrafish and urodele amphibians 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 transient expression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM) transcription factors. Here, we utilized an OSKM-encoding non-integrating vector to induce transient reprogramming of mammalian cardiomyocytes in vitro. Reprogramming factor expression in neonatal rat cardiomyocytes triggered rapid cell dedifferentiation characterized by downregulation of cardiomyocyte specific gene and protein expression, sarcomere dis-assembly and loss of autorhythmic contractile activity. Concomitantly, a significant increase in cell cycle related gene expression and Ki67 positive cells was observed, indicating that dedifferentiated cardiomyocytes possess an enhanced proliferative capacity. A small proportion of cardiomyocytes progressed through mesenchymal to epithelial transition, further indicating the initiation of cell reprogramming. However, complete reprogramming to a pluripotent-like state was not achieved for the duration of the study (20 days), both in standard and embryonic stem cell culture media conditions. The transient nature of this partial reprogramming response was confirmed as cardiomyocyte-specific cell morphology, gene expression and contractile activity were recovered by day 15 after viral transduction. Further investigations into the complete downstream biological effects of ectopic OSKM expression in cardiomyocytes and the fate of these reprogrammed cells are warranted. Our results to date suggest that transient reprogramming could be a feasible strategy to recapitulate regenerative mechanisms of lower vertebrates and inform direct gene therapy approaches to cardiac regenerative medicine.

cell biology↗

bioRxiv2019

TNAP limits TGF-β-dependent cardiac and skeletal muscle fibrosis by inactivating 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 beta (TGF-{beta}) plays a pivotal role in fibrosis, activating pro-fibrotic gene programs via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2/3 have remained poorly characterized. Here we show that tissue non-specific alkaline phosphatase (TNAP) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and the abrogation of TGF-{beta} signalling in TNAP positive cells reduces vascular and interstitial fibrosis. We show that TNAP co-localizes and interacts with SMAD2. 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-{beta} signalling and likely represents a mechanism to limit fibrosis.\n\nSummary statementThis paper shows that tissue non-specific alkaline phosphatase negatively regulates TGF-{beta} signalling and may represent a mechanism to limit fibrosis through SMAD dephosphorylation.

cell biology↗

bioRxiv2017

MCAM controls cell autonomous polarity in myogenic and chondrogenic differentiation

Cell polarity has a fundamental role in shaping the morphology of cells and growing tissues. Polarity is commonly thought to be established in response to extracellular signals. Here we used a minimal in vitro assay that enabled us to monitor the determination of cell polarity in myogenic and chondrogenic differentiation in the absence of external signalling gradients. We demonstrate that the initiation of cell polarity is regulated by melanoma cell adhesion molecule (MCAM). We found highly polarized localization of MCAM, Moesin (MSN), Scribble (SCRIB) and Van-Gogh-like 2 (VANGL2) at the distal end of elongating myotubes. Knockout of MCAM or elimination of its endocytosis motif does not impair the initiation of myogenesis or myoblast fusion, but prevents myotube elongation. MSN, SCRIB and VANGL2 remain uniformly distributed in MCAM knockout cells. We show that MCAM is also required at early stages of chondrogenic differentiation. In both myogenic and chondrogenic differentiation MCAM knockout leads to transcriptional downregulation of Scrib and enhanced MAP kinase activity. Our data demonstrates the importance of cell autonomous polarity in differentiation.

cell biology↗

bioRxiv2017

Non-viral induction of transient cell reprogramming in skeletal muscle to enhance tissue regeneration

Somatic cells can be reprogrammed to pluripotency in vivo by overexpression of defined transcription factors. While their sustained expression triggers tumorigenesis, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratoma formation. We sought to achieve transient reprogramming within mouse skeletal muscle with a localized injection of plasmid DNA (pDNA) and hypothesized that this would enhance regeneration after severe injury. Intramuscular administration of reprogramming pDNA rapidly upregulated pluripotency (Nanog, Ecat1, Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various mouse strains. Mononucleated cells expressing such markers appeared promptly in clusters among myofibers, but proliferated only transiently and did not lead to the generation of teratomas. Nanog was also upregulated in the gastrocnemius when reprogramming factors were administered 7 days after laceration of its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centro-nucleated myofibers and reduced fibrosis. These results suggest that in vivo transient reprogramming may constitute a novel strategy towards the acceleration of regeneration following muscle injury, based on the induction of transiently-proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted.

developmental biology↗