Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Surgical oncological emergencies in octogenarian patients.

Background: Surgical oncological emergencies represent a frequent challenge in acute settings, with postoperative courses characterized by high morbidity and mortality. An accurate selection of patients who could benefit from surgery is essential to avoid unnecessary invasive treatment. In this study, we tried to determine if advanced age (>80 years) represents a risk factor for negative short-term outcome in patients undergoing emergency surgery for acute abdominal oncological illness. Methods: We retrospectively analyzed the records of patients who underwent emergency oncological surgery at the Department of Acute Care Surgery of Padua General Hospital from January 2018 to December 2022. One hundred two cancer patients were included in the study. Among them, 42 were aged ≥80 years (41%). Multiple preoperative and postoperative parameters were recorded, and the follow-up period was at least 90 days. Multivariate logistic regression analyses were used to identify factors associated with short-term postoperative outcomes. Results: In the octogenarian group, 30-day mortality was 11% vs. 9.5% in the younger group [p = not significant (ns)] and 90-day mortality was 17.6% in the octogenarian group vs. 20.5% in the younger group (p = ns). Postoperative morbidity and hospital length of stay were not significantly different in the two groups. Low albumin levels [odds ratio (OR) 30.6, 9.51-87.07] and elevated lactate dehydrogenase (LDH) levels (OR 26.4, 9.18-75.83) were predictive for short-term mortality in surgical oncological emergencies. Conclusion: Advanced age is not a risk factor for negative outcomes in surgical oncological emergencies. Therefore, surgical options should be considered in octogenarians with oncological emergencies and acceptable clinical conditions. Serum albumin levels and LDH can help predict the postoperative outcome after surgery for oncological emergencies.

Aberrant axon initial segment plasticity and intrinsic excitability of ALS hiPSC motor neurons.

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons.

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds.

Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image (HCI) analysis. This study describes the development of a scalable human induced pluripotent stem cell (iPSC)-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates. In this platform, optogenetic stimulation of the motor neurons elicits robust myofiber-contractions, providing a functional readout of neuromuscular transmission. Additionally, HCI analysis provides rapid and automated quantification of axonal outgrowth, myofiber morphology, and neuromuscular synapse number and morphology. By incorporating amyotrophic lateral sclerosis (ALS)-related TDP-43G298Smutant motor neurons and CRISPR-corrected controls, key neuromuscular disease phenotypes are recapitulated, including weaker myofiber contractions, reduced axonal outgrowth, and reduced number of neuromuscular synapses. Treatment with a candidate ALS drug, the receptor-interacting protein kinase-1 (RIPK1)-inhibitor necrostatin-1, rescues these phenotypes in a dose-dependent manner, highlighting the potential of this platform to screen novel treatments for neuromuscular diseases.

Cardiovascular complications of diabetes: role of non-coding RNAs in the crosstalk between immune and cardiovascular systems.

Diabetes mellitus, a group of metabolic disorders characterized by high levels of blood glucose caused by insulin defect or impairment, is a major risk factor for cardiovascular diseases and related mortality. Patients with diabetes experience a state of chronic or intermittent hyperglycemia resulting in damage to the vasculature, leading to micro- and macro-vascular diseases. These conditions are associated with low-grade chronic inflammation and accelerated atherosclerosis. Several classes of leukocytes have been implicated in diabetic cardiovascular impairment. Although the molecular pathways through which diabetes elicits an inflammatory response have attracted significant attention, how they contribute to altering cardiovascular homeostasis is still incompletely understood. In this respect, non-coding RNAs (ncRNAs) are a still largely under-investigated class of transcripts that may play a fundamental role. This review article gathers the current knowledge on the function of ncRNAs in the crosstalk between immune and cardiovascular cells in the context of diabetic complications, highlighting the influence of biological sex in such mechanisms and exploring the potential role of ncRNAs as biomarkers and targets for treatments. The discussion closes by offering an overview of the ncRNAs involved in the increased cardiovascular risk suffered by patients with diabetes facing Sars-CoV-2 infection.

Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter.

V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Laparoscopic Heller-Dor is an effective long-term treatment for end-stage achalasia.

BACKGROUND: The end-stage achalasia is a difficult condition to treat, for the esophageal diameter and conformation of the gullet, that may progress to a sigmoid shape. The aim of this study was to examine the outcome of Laparoscopic Heller-Dor in patients with end-stage achalasia, comparing them with patients who had mega-esophagus without a sigmoid shape. METHODS: From 1992 to 2020, patients with a diagnosis of sigmoid esophagus, or radiological stage IV achalasia (the SE group), and patients with a straight esophagus larger than 6 cm in diameter, or radiological stage III achalasia (the NSE group), were all treated with LHD. The two groups were compared in terms of patients' symptoms, based on the Eckardt score, and on barium swallow, endoscopy and manometry performed before and after the treatment. The failure of the treatment was defined as an Eckardt score > 3, or the need for further treatment. RESULTS: The study involved 164 patients: 73 in the SE group and 91 in the NSE group. No intra- or postoperative mortality was recorded. The median follow-up was 51 months (IQR 25-107). The outcome was satisfactory in 71.2% of patients in the SE group, and in 89% of those in the NSE group (p = 0.005). CONCLUSIONS: SE is certainly the worst condition of the disease and the final outcome of LHD, in term of symptom control, is inferior compared to NSE. Despite this, almost 3/4 of the SE patients experienced a significant relieve in symptoms after LHD, which may therefore still be the first surgical option to offer to these patients, before considering esophagectomy.

Results of a prospective observational study of autologous peripheral blood mononuclear cell therapy for no-option critical limb-threatening ischemia and severe diabetic foot ulcers.

BACKGROUND: Cell therapy with autologous peripheral blood mononuclear cells (PB-MNCs) may help restore limb perfusion in patients with diabetes mellitus and critical limb-threatening ischemia (CLTI) deemed not eligible for revascularization procedures and consequently at risk for major amputation (no-option). Fundamental is to establish its clinical value and to identify candidates with a greater benefit over time. Assessing the frequency of PB circulating angiogenic cells and extracellular vesicles (EVs) may help in guiding candidate selection. METHODS: We conducted a prospective, non-controlled, observational study on no-option CLTI diabetic patients that underwent intramuscular PB-MNCs therapy, which consisted of more cell treatments repeated a maximum of three times. The primary endpoint was amputation rate at 1 year following the first treatment with PB-MNCs. We evaluated ulcer healing, walking capability, and mortality during the follow-up period. We assessed angiogenic cells and EVs at baseline and after each cell treatment, according to primary outcome and tissue perfusion at the last treatment [measured as transcutaneous oxygen pressure (TcPO2)]. RESULTS: 50 patients were consecutively enrolled and the primary endpoint was 16%. TcPO2 increased after PB-MNCs therapy (17.2 ± 11.6 vs 39.1 ± 21.8 mmHg, p < .0001), and ulcers healed with back-to-walk were observed in 60% of the study population (88% of survivors) during follow-up (median 1.5 years). Patients with a high level of TcPO2 (≥ 40 mmHg) after the last treatment showed a high frequency of small EVs at enrollment. CONCLUSIONS: In no-option CLTI diabetic patients, PB-MNCs therapy led to an improvement in tissue perfusion, a high rate of healing, and back-to-walk. Coupling circulating cellular markers of angiogenesis could help in the identification of patients with a better clinical benefit over time.

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

Biobased Elastomer Nanofibers Guide Light-Controlled Human-iPSC-Derived Skeletal Myofibers.

Generating skeletal muscle tissue that mimics the cellular alignment, maturation, and function of native skeletal muscle is an ongoing challenge in disease modeling and regenerative therapies. Skeletal muscle cultures require extracellular guidance and mechanical support to stabilize contractile myofibers. Existing microfabrication-based solutions are limited by complex fabrication steps, low throughput, and challenges in measuring dynamic contractile function. Here, the synthesis and characterization of a new biobased nanohybrid elastomer, which is electrospun into aligned nanofiber sheets to mimic the skeletal muscle extracellular matrix, is presented. The polymer exhibits remarkable hyperelasticity well-matched to that of native skeletal muscle (≈11-50 kPa), with ultimate strain ≈1000%, and elastic modulus ≈25 kPa. Uniaxially aligned nanofibers guide myoblast alignment, enhance sarcomere formation, and promote a ≈32% increase in myotube fusion and ≈50% increase in myofiber maturation. The elastomer nanofibers stabilize optogenetically controlled human induced pluripotent stem cell derived skeletal myofibers. When activated by blue light, the myofiber-nanofiber hybrid constructs maintain a significantly higher (>200%) contraction velocity and specific force (>280%) compared to conventional culture methods. The engineered myofibers exhibit a power density of ≈35 W m-3 . This system is a promising new skeletal muscle tissue model for applications in muscular disease modeling, drug discovery, and muscle regeneration.

Esophageal Achalasia: Diagnostic Evaluation.

A precise diagnosis is key to the successful treatment of achalasia. Barium swallow, upper endoscopy and high-resolution manometry provide the necessary information about a patient's anatomy, absence of other diseases, and type of achalasia (I, II, III). High-resolution manometry also has prognostic value, the best results of treatment being obtained in type II achalasia according to the Chicago classification. Abdominal CT scanning and endoscopic ultrasound might be warranted if an underlying malignancy is suspected.

Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines.

Repurposing of drugs for new therapeutic use has received considerable attention for its potential to limit time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to promote a desired phenotype. We used data from the Connectivity Map (CMap) to produce a ranked list of drugs according to their potential to activate transcription factors that mediate myeloid differentiation of leukemic progenitor cells. To validate our strategy, we tested the in vitro differentiation potential of candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, which were ranked high in the inferred list, were confirmed to promote significant differentiation of HL-60. These compounds may be considered candidate for differentiation therapy. The method that we have developed is versatile and it can be adapted to different drug repurposing projects.

Characterization of the Skeletal Muscle Secretome Reveals a Role for Extracellular Vesicles and IL1α/IL1ß in Restricting Fibro/Adipogenic Progenitor Adipogenesis.

Repeated mechanical stress causes injuries in the adult skeletal muscle that need to be repaired. Although muscle regeneration is a highly efficient process, it fails in some pathological conditions, compromising tissue functionality. This may be caused by aberrant cell-cell communication, resulting in the deposition of fibrotic and adipose infiltrates. Here, we investigate in vivo changes in the profile of skeletal muscle secretome during the regeneration process to suggest new targetable regulatory circuits whose failure may lead to tissue degeneration in pathological conditions. We describe the kinetic variation of expression levels of 76 secreted proteins during the regeneration process. In addition, we profile the gene expression of immune cells, endothelial cells, satellite cells, and fibro-adipogenic progenitors. This analysis allowed us to annotate each cell-type with the cytokines and receptors they have the potential to synthetize, thus making it possible to draw a cell-cell interaction map. We next selected 12 cytokines whose receptors are expressed in FAPs and tested their ability to modulate FAP adipogenesis and proliferation. We observed that IL1α and IL1ß potently inhibit FAP adipogenesis, while EGF and BTC notably promote FAP proliferation. In addition, we characterized the cross-talk mediated by extracellular vesicles (EVs). We first monitored the modulation of muscle EV cargo during tissue regeneration. Using a single-vesicle flow cytometry approach, we observed that EVs differentially affect the uptake of RNA and proteins into their lumen. We also investigated the EV capability to interact with SCs and FAPs and to modulate their proliferation and differentiation. We conclude that both cytokines and EVs secreted during muscle regeneration have the potential to modulate adipogenic differentiation of FAPs. The results of our approach provide a system-wide picture of mechanisms that control cell fate during the regeneration process in the muscle niche.

A Resource for the Network Representation of Cell Perturbations Caused by SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has caused more than 2.3 million casualties worldwide and the lack of effective treatments is a major health concern. The development of targeted drugs is held back due to a limited understanding of the molecular mechanisms underlying the perturbation of cell physiology observed after viral infection. Recently, several approaches, aimed at identifying cellular proteins that may contribute to COVID-19 pathology, have been reported. Albeit valuable, this information offers limited mechanistic insight as these efforts have produced long lists of cellular proteins, the majority of which are not annotated to any cellular pathway. We have embarked in a project aimed at bridging this mechanistic gap by developing a new bioinformatic approach to estimate the functional distance between a subset of proteins and a list of pathways. A comprehensive literature search allowed us to annotate, in the SIGNOR 2.0 resource, causal information underlying the main molecular mechanisms through which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses affect the host-cell physiology. Next, we developed a new strategy that enabled us to link SARS-CoV-2 interacting proteins to cellular phenotypes via paths of causal relationships. Remarkably, the extensive information about inhibitors of signaling proteins annotated in SIGNOR 2.0 makes it possible to formulate new potential therapeutic strategies. The proposed approach, which is generally applicable, generated a literature-based causal network that can be used as a framework to formulate informed mechanistic hypotheses on COVID-19 etiology and pathology.

Skeletal Muscle Subpopulation Rearrangements upon Rhabdomyosarcoma Development through Single-Cell Mass Cytometry.

The embryonal rhabdomyosarcoma (eRMS) is a soft tissue sarcoma commonly affecting the head and neck, the extremities and the genitourinary tract. To contribute to revealing the cell types that may originate this tumor, we exploited mass cytometry, a single-cell technique that, by using heavy-metal-tagged antibodies, allows the accurate monitoring of the changes occurring in the mononuclear cell composition of skeletal muscle tissue during tumor development. To this end, we compared cell populations of healthy muscles with those from spatiotemporal-induced eRMS tumors in a mouse model (LSL-KrasG12D/+;Tp53Fl/Fl) that can be used to develop rhabdomyosarcoma by means of infection with an adenovirus vector expressing Cre (Ad-Cre) recombinase. By monitoring different time points after tumor induction, we were able to analyze tumor progression and composition, identifying fibro/adipogenic progenitors (FAPs) as the cell type that, in this model system, had a pivotal role in tumor development. In vitro studies highlighted that both FAPs and satellite cells (SCs), upon infection with the Ad-Cre, acquired the potential to develop rhabdomyosarcomas when transplanted into immunocompromised mice. However, only infected FAPs had an antigen profile that was similar to embryonal rhabdomyosarcoma cells. Overall, our analysis supports the involvement of FAPs in eRMS development.

SCA-1 micro-heterogeneity in the fate decision of dystrophic fibro/adipogenic progenitors.

The term micro-heterogeneity refers to non-genetic cell to cell variability observed in a bell-shaped distribution of the expression of a trait within a population. The contribution of micro-heterogeneity to physiology and pathology remains largely uncharacterised. To address such an issue, we investigated the impact of heterogeneity in skeletal muscle fibro/adipogenic progenitors (FAPs) isolated from an animal model of Duchenne muscular dystrophy (DMD), the mdx mouse. FAPs play an essential role in muscle homoeostasis. However, in pathological conditions or ageing, they are the source of intramuscular infiltrations of fibrotic or adipose tissue. By applying a multiplex flow cytometry assay, we characterised and purified from mdx muscles two FAP cell states expressing different levels of SCA-1. The two cell states are morphologically identical and repopulate each other after several growth cycles. However, they differ in their in vitro behaviour. Cells expressing higher levels of SCA-1 (SCA1-High-FAPs) differentiate more readily into adipocytes while, when exposed to a fibrogenic stimulation, increase the expression of Col1a1 and Timp1 mRNA. A transcriptomic analysis confirmed the adipogenic propensity of SCA1-High-FAPs. In addition, SCA1-High-FAPs proliferate more extensively ex vivo and display more proliferating cells in dystrophic muscles in comparison to SCA1-Low-FAPs. Adipogenesis of both FAP cell states is inhibited in vitro by leucocytes from young dystrophic mice, while leucocytes isolated from aged dystrophic mice are less effective in limiting the adipogenesis of SCA1-High-FAPs suggesting a differential regulatory effect of the microenvironment on micro-heterogeneity. Our data suggest that FAP micro-heterogeneity is modulated in pathological conditions and that this heterogeneity in turn may impact on the behaviour of interstitial mesenchymal cells in genetic diseases.

When a Friend Becomes Your Enemy: Natural Killer Cells in Atherosclerosis and Atherosclerosis-Associated Risk Factors.

Atherosclerosis (ATS), the change in structure and function of arteries with associated lesion formation and altered blood flow, is the leading cause of cardiovascular disease, the number one killer worldwide. Beyond dyslipidemia, chronic inflammation, together with aberrant phenotype and function of cells of both the innate and adaptive immune system, are now recognized as relevant contributors to atherosclerosis onset and progression. While the role of macrophages and T cells in atherosclerosis has been addressed in several studies, Natural Killer cells (NKs) represent a poorly explored immune cell type, that deserves attention, due to NKs' emerging contribution to vascular homeostasis. Furthermore, the possibility to re-polarize the immune system has emerged as a relevant tool to design new therapies, with some succesfull exmples in the field of cancer immunotherapy. Thus, a deeper knowledge of NK cell pathophysiology in the context of atherosclerosis and atherosclerosis-associated risk factors could help developing new preventive and treatment strategies, and decipher the complex scenario/history from "the risk factors for atherosclerosis" Here, we review the current knowledge about NK cell phenotype and activities in atherosclerosis and selected atherosclerosis risk factors, namely type-2 diabetes and obesity, and discuss the related NK-cell oriented environmental signals.

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy.