Biology subjects

Sampaolesi, M.

Publications and source records attributed to Sampaolesi, M..

10 recordsLinked to original sources

bioRxiv2025

Pde5a Deficiency Prevents Diet-Induced Obesity via Adipose cAMP-PKA Activation Enhancing Fat Browning

Cyclic nucleotides are critical regulators of adaptive thermogenesis and adipogenesis, with their intracellular levels finely tuned by phosphodiesterases. Phosphodiesterase type 5 (PDE5A) modulates cyclic guanosine monophosphate levels in adipocytes. While PDE5A inhibition has shown promise in patients with diabetes, its role in metabolism remains unclear. Using Pde5a knockout mouse models, we demonstrated that mice lacking Pde5a exhibit enhanced browning of white adipose tissue and reduced hepatic fat content. Following high-fat diet, Pde5a-/- mice are resistant to obesity, displaying improved glucose metabolism and enhanced thermogenesis. These protective effects stem from an early developmental knockdown of Pde5a, leading to a metabolic reprogramming driven by cAMP-PKA pathway activation. The convergence of cGMP and cAMP signaling orchestrates thermogenic and systemic metabolic adaptations. Our findings establish PDE5A as a novel regulator of energy homeostasis, suggesting its inhibition as a valuable adjuvant therapy for metabolic disorders.

cell biology↗

bioRxiv2023

Adult stem cell characterization from the Medial Gastrocnemius and Semitendinosus muscles in early development of cerebral palsy pathology

Cerebral palsy (CP) is one of the most common lifelong conditions leading to childhood physical disability. Literature reported previously altered muscle properties such as lower number of satellite cells (SCs), with altered fusion capacity. However, these observations highly vary among studies, possibly due to heterogeneity in patient population, lack of appropriate control data, methodology and different assessed muscle. In this study we aimed to strengthen previous observations and to understand the heterogeneity of CP muscle pathology. Myogenic differentiation of SCs from the Medial Gastrocnemius (MG) muscle of patients with CP (n=16, 3-9 years old) showed higher fusion capacity compared to age-matched typically developing children (TD, n=13). Furthermore, we uniquely assessed cells of two different lower limb muscles and showed a decreased myogenic potency in cells from the Semitendinosus (ST) compared to the MG. Longitudinal assessments, one year after the first botulinum toxin treatment, showed slightly reduced SC representations and lower fusion capacity. Finally, we proved the robustness of our data, by assessing in parallel the myogenic capacity of two samples from the same TD muscle. In conclusion, these data confirmed previous findings of increased SC fusion capacity from MG muscle of young patients with CP compared to age-matched TD. Further elaboration is reported on potential factors contributing to heterogeneity, such as assessed muscle, CP progression and reliability of primary outcome parameters.

developmental biology↗

bioRxiv2022

Long-Term Culture of Patient-Derived Cardiac Organoids Recapitulated Duchenne Muscular Dystrophy Cardiomyopathy and Disease Progression

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to-date incurable. The major cause of death is dilated cardiomyopathy, however the pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived pluripotent stem cells (DMD-CO) and isogenic-corrected controls (DMD-Iso-CO) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-CO lacks initial proliferative capacity, displayed a progressive loss of -sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, the cardiomyocyte deteriorated over time, and fibrosis and adipogenesis were observed in DMD-CO. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-CO which were associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.

cell biology↗

bioRxiv2022

Cardiomyocyte differentiation from iPS 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 decisions and autophagy. Bcl-2 acts by both direct interaction with different components of the pathways involved and by intervening in intracellular Ca2+ signalling. The function of Bcl-2 is in turn regulated by post-translational modifications including phosphorylation at different sites by various protein kinases. Besides functions in cell death and apoptosis, Bcl-2 regulates cell differentiation processes, including of cardiomyocytes, although its mechanism of action in this process is not fully elucidated. To further address the role of Bcl-2 in cardiomyocyte differentiation, we investigated the effect of its genetic knockout by CRISPR/Cas9 on the differentiation and functional maturation trajectory of human induced pluripotent stem cells (hiPSC) to cardiomyocytes. Our results indicate that differentiation of hiPSC to cardiomyocytes is delayed by Bcl-2 KO. This effect was associated with smaller areas of spontaneously beating cells. At the cell population level this exhibited in reduced expression and activity of the cardiomyocyte Ca2+ toolkit. However, when restricting the comparison to the active areas exclusively Bcl-2 KO did not show to significantly alter the functionality of the differentiated cardiomyocytes. Finally, Bcl-2 KO reduced c-Myc expression early in the cardiac differentiation process which may account at least in part for the here observed delay in cardiac differentiation resulting reduced efficiency of the differentiation process. These data are supportive of a pivotal role for Bcl-2 in cardiomyocyte differentiation and maturation.

developmental biology↗

bioRxiv2021

PGC-1α in the myofibers regulates the balance between myogenic and adipogenic progenitors affecting muscle regeneration

Skeletal muscle repair is accomplished by satellite cells (MuSC) in cooperation with interstitial stromal cells (ISCs). So far, the relationship between the function of these cells and the metabolic state of myofibers remains unclear. The present study reports alterations in the proportion of both MuSCs and adipogenesis regulators (Aregs) induced by overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1) in the myofibers (MCK-PGC-1 mice). Although PGC-1-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1 mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from WT donors. Moreover, both young and aged MCK-PGC-1 animals show reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. These results provide new insights on the role played by PGC-1 in promoting myogenesis and hindering adipogenesis in the skeletal muscle.

molecular biology↗

bioRxiv2021

Human iPSC-Based Model Reveals NOX4 as Therapeutic Target in Duchenne Cardiomyopathy

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder, caused by mutations in the Dystrophin gene. Cardiomyopathy is one of the major causes of early death. In this study, we used DMD patient-specific induced pluripotent stem cells (iPSCs) to model cardiomyopathic features in DMD and unravel novel pathological mechanistic insights. Cardiomyocytes (CMs) differentiated from DMD iPSCs showed enhanced premature cell death, due to significantly elevated intracellular reactive oxygen species (ROS) concentrations, as a result of depolarized mitochondria and high NADPH oxidase 4 (NOX4) protein levels. Genetic correction of Dystrophin through CRISPR/Cas9 editing restored normal ROS levels. Application of ROS reduction by N-acetyl-L-cysteine (NAC), partial Dystrophin re-expression by ataluren (PTC124) and enhancing mitochondrial electron transport chain function by idebenone improved cell survival of DMD iPSC-CMs. We show applications that could counteract the detrimental oxidative stress environment in DMD iPSC-CMs by stimulating adenosine triphosphate (ATP) production. ATP could bind to the ATP-binding domain in the NOX4 enzyme, and we demonstrate that ATP resulted in partial inhibition of the NADPH-dependent ROS production of NOX4. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose to target ROS production and prevent the detrimental effects of NOX4 on DMD CMs as a promising therapeutic strategy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/460090v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d3606borg.highwire.dtl.DTLVardef@15f1d88org.highwire.dtl.DTLVardef@18bfb9corg.highwire.dtl.DTLVardef@133c6bd_HPS_FORMAT_FIGEXP M_FIG The use of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from Duchenne muscular dystrophy (DMD) patients to model cardiomyopathic features in DMD and unravel novel pathological mechanistic insights. DMD iPSC-CMs showed accelerated cell death, caused by increased intracellular reactive oxygen species (ROS) levels. By intervention at different target sites, beneficial effects on the mitochondrial membrane potential ({Delta}{Psi}m) and the expression and ROS-producing activity of the cardiac-specific NADPH-oxidase 4 isoform (NOX4) were observed, resulting in an increased cell survival and function of DMD iPSC-CMs. C_FIG

developmental biology↗

bioRxiv2020

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

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 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 without compromising their ability to differentiate towards cardiomyocytes. This effect is mediated through the activation of the Notch signaling pathway. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents, which opens the door to new therapeutic solutions in the treatment of muscular dystrophy.

cell biology↗

bioRxiv2020

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

Neuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

neuroscience↗

bioRxiv2020

Actuation Enhances Patterning in Human Neural Tube Organoids

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.

bioengineering↗

bioRxiv2019

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

Defective fibroblast migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective treatment able to improve wound healing capacity. However, the molecular mechanisms connecting their beneficial outcomes with the wound healing process are still unrevealed. Here, we show that AMG modulates primary fibroblast migration and accelerates skin re-epithelialization without affecting cell proliferation. We demonstrate that AMG is enriched in a pool of WH-associated growth factors that may provide the initiation signal for a faster endogenous wound healing response. This, in turn leads to increased cell migration rate by elevating activity of extracellular signal-regulated kinase (ERK) pathway and subsequent activation of matrix metalloproteinase expression and their extracellular enzymatic activity. Moreover, AMG-treated wounds showed increased granulation tissue formation and organized collagen content. Overall, we shed light on AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process.

molecular biology↗