Lineage Plasticity and Regenerative Potential of Adult Muscle Stem Cells: Investigation of Satellite Cell Direct-Reprogramming and Pericyte Self-Renewal

Satellite cells are responsible for most of adult skeletal muscle regeneration. Upon activation they differentiate into transient amplifying myoblasts that undergo cell fusion to form multinucleated fibres. Despite their remarkable differentiation ability and the positive outcomes obtained with transplantation in dystrophic mice and recently in patients with oculo-pharyngeal muscular dystrophy (OPMD), clinical trials in patients with Duchenne muscular dystrophy (DMD) showed limited efficacy, mainly ascribed to myoblasts low survival and poor migration ability. Muscle pericyte-derived mesoangioblasts (perivascular cells associated to the capillaries) also contribute to muscle regeneration and colonise the satellite cell niche. These cells can be injected systemically and migrate through the vascular endothelium, circumventing the necessity of multiple intra-muscular injections. Mesoangioblasts have been also tested in a recently completed phase I / II clinical trial to assess their safety profile in five DMD patients (EudraCT no. 2011-000176-33). We hypothesise that exploiting the key properties of myoblasts and mesoangioblasts may have the potential to produce clinically relevant cells, superior to those currently available. This work shows that exposure to molecules involved in pericyte specification such as the Notch ligand DLL4 and the growth factor PDGF-BB can induce direct reprogramming of primary satellite cells to pericyte-like cells. Reprogrammed cells acquire perivascular marker expression without losing the satellite cell marker Pax7. These highly myogenic cells can be expanded in culture and showed increased engraftment. In vitro and in vivo experiments also showed improved migration ability, similar to what has been observed with mesoangioblasts. Additionally, this thesis includes a set of experiments aiming to assess the self-renewal potential of mesoangioblast-derived cells via serial transplantation assays. Overall, the results obtained improve our understanding of smooth / skeletal fate choice and self-renewal, providing evidence of the possibility of exploiting a direct reprogramming approach to allow systemic delivery of myoblasts for cell therapies of muscular dystrophies

Primary human organoids models: Current progress and key milestones

During the past 10 years the world has experienced enormous progress in the organoids field. Human organoids have shown huge potential to study organ development, homeostasis and to model diseases in vitro. The organoid technology has been widely and increasingly applied to generate patient-specific in vitro 3D cultures, starting from both primary and reprogrammed stem/progenitor cells. This has consequently fostered the development of innovative disease models and new regenerative therapies. Human primary, or adult stem/progenitor cell-derived, organoids can be derived from both healthy and pathological primary tissue samples spanning from fetal to adult age. The resulting 3D culture can be maintained for several months and even years, while retaining and resembling its original tissue’s properties. As the potential of this technology expands, new approaches are emerging to further improve organoid applications in biology and medicine. This review discusses the main organs and tissues which, as of today, have been modelled in vitro using primary organoid culture systems. Moreover, we also discuss the advantages, limitations, and future perspectives of primary human organoids in the fields of developmental biology, disease modelling, drug testing and regenerative medicine

Edge-Illumination X-Ray Dark-Field Tomography

Dark-field imaging is an x-ray technique used to highlight subpixel, typically micrometer-scale, density fluctuations. It is often used alongside standard attenuation-based and also phase-contrast x-ray imaging, which both see regular use in tomography. We present x-ray dark-field computed tomography (CT) with a laboratory edge-illumination setup. The dark-field contrast is shown to increase linearly with the x-ray path length through the imaged object, a prerequisite for the use of standard tomographic reconstruction approaches. A multimaterial, custom-built phantom is used to show how dark-field contrast CT can complement attenuation contrast CT for the separation of materials based on their microstructure. As an example of a more complex, biological sample, we present a model rat heart. We show, by comparison with attenuation contrast tomography, that dark-field enables the identification of additional structures undetected through the attenuation contrast channel, as well as offering a consistently sharper reconstructed image

Decellularized skeletal muscles display neurotrophic effects in three‐dimensional organotypic cultures

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three‐dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required

Decellularized skeletal muscles support the generation of in vitro neuromuscular tissue models

Decellularized skeletal muscle (dSkM) constructs have received much attention in recent years due to the versatility of their applications in vitro. In search of adequate in vitro models of the skeletal muscle tissue, the dSkM offers great advantages in terms of the preservation of native-tissue complexity, including three-dimensional organization, the presence of residual signaling molecules within the construct, and their myogenic and neurotrophic abilities. Here, we attempted to develop a 3D model of neuromuscular tissue. To do so, we repopulated rat dSkM with human primary myogenic cells along with murine fibroblasts and we coupled them with organotypic rat spinal cord samples. Such culture conditions not only maintained multiple cell type viability in a long-term experimental setup, but also resulted in functionally active construct capable of contraction. In addition, we have developed a customized culture system which enabled easy access, imaging, and analysis of in vitro engineered co-cultures. This work demonstrates the ability of dSkM to support the development of a contractile 3D in vitro model of neuromuscular tissue fit for long-term experimental evaluations

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

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types

RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner.

Nasopharyngeal carcinoma (NPC) is a highly aggressive tumor characterized by distant metastasis. Deletion or down-regulation of the tumor suppressor protein ras-association domain family protein1 isoform A (RASSF1A) has been confirmed to be a key event in NPC progression; however, little is known about the effects or underlying mechanism of RASSF1A on the malignant phenotype. In the present study, we observed that RASSF1A expression inhibited the malignant phenotypes of NPC cells. Stable silencing of RASSF1A in NPC cell lines induced self-renewal properties and tumorigenicity in vivo/in vitro and the acquisition of an invasive phenotype in vitro. Mechanistically, RASSF1A inactivated Yes-associated Protein 1 (YAP1), a transcriptional coactivator, through actin remodeling, which further contributed to Platelet Derived Growth Factor Subunit B (PDGFB) transcription inhibition. Treatment with ectopic PDGFB partially increased the malignancy of NPC cells with transient knockdown of YAP1. Collectively, these findings suggest that RASSF1A inhibits malignant phenotypes by repressing PDGFB expression in a YAP1-dependent manner. PDGFB may serve as a potential interest of therapeutic regulators in patients with metastatic NPC