State of the Art Procedures for the Isolation and Characterization of Mesoangioblasts.

Adult skeletal muscle is a dynamic tissue able to regenerate quite efficiently, thanks to the presence of stem cell machinery. Besides the quiescent satellite cells that are activated upon injury or paracrine factors, other stem cells are described to be directly or indirectly involved in adult myogenesis. Mesoangioblasts (MABs) are vessel-associated stem cells originally isolated from embryonic dorsal aorta and, at later stages, from the adult muscle interstitium expressing pericyte markers. Adult MABs entered clinical trials for the treatment of Duchenne muscular dystrophy and the transcriptome of human fetal MABs has been described. In addition, single cell RNA-seq analyses provide novel information on adult murine MABs and more in general in interstitial muscle stem cells. This chapter provides state-of-the-art techniques to isolate and characterize murine MABs, fetal and adult human MABs.

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

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 is incurable. The major cause of death is dilated cardiomyopathy however, its 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 induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) 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-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was 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.

Upregulation of miR181a/miR212 Improves Myogenic Commitment in Murine Fusion-Negative Rhabdomyosarcoma.

Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs in vivo by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation in vivo as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications.

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 human 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 that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

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.

MICAL2 is essential for myogenic lineage commitment.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

Hair EtG: Alterations in segment levels accompanying hair growth.

Hair levels of the direct ethanol biomarker ethyl glucuronide (EtG) are used to evaluate history of alcohol intake. The proximal 3 cm of the hair sample is most often analyzed and this is assumed to represent the intake of ethanol over approximately the past three months. The aim of this study was to investigate change of EtG levels during hair growth in an ethanol-abstinent period. Twenty-seven patients were recruited from an alcohol treatment clinic. A hair sample was collected after hospitalization and EtG was analyzed in the 0-3 cm hair segment (T1). Another hair sample was collected after one month of abstinence and EtG was analyzed in the 1-4 cm hair segment (T2), discarding the proximal 1 cm (0-1 cm) of the segment. As a result of the segment choice and assuming a hair growth rate of 1 cm per month, T2 should represent roughly the same time of alcohol exposure as segment T1. The median concentration of EtG in T1 was 100 pg/mg (range 7.7-1320) and the median concentration in T2 was 53.4 pg/mg (range < LOQ-692). EtG concentrations decreased significantly from T1 to T2 (p = 0.003) and the median change in EtG from T1 to T2 was -46.0%. This study shows decreasing EtG concentrations in most subjects in a hair segment during growth when comparing two segments that is assumed to represent roughly the same period of alcohol intake. Further research is needed to confirm if this observed decline of EtG is a result of wash-out-effects of EtG or other factors.

Evaluation of Poly(Lactic-co-glycolic) Acid Alone or in Combination with Hydroxyapatite on Human-Periosteal Cells Bone Differentiation and in Sinus Lift Treatment.

Most recent advances in tissue engineering in the fields of oral surgery and dentistry have aimed to restore hard and soft tissues. Further improvement of these therapies may involve more biological approaches and the use of dental tissue stem cells in combination with inorganic/organic scaffolds. In this study, we analyzed the osteoconductivity of two different inorganic scaffolds based on poly (lactic-co-glycolic) acid alone (PLGA-Fisiograft) or in combination with hydroxyapatite (PLGA/HA-Alos) in comparison with an organic material based on equine collagen (PARASORB Sombrero) both in vitro and in vivo. We developed a simple in vitro model in which periosteum-derived stem cells were grown in contact with chips of these scaffolds to mimic bone mineralization. The viability of cells and material osteoconductivity were evaluated by osteogenic gene expression and histological analyses at different time points. In addition, the capacity of scaffolds to improve bone healing in sinus lift was examined. Our results demonstrated that the osteoconductivity of PLGA/HA-Alos and the efficacy of scaffolds in promoting bone healing in the sinus lift were increased. Thus, new clinical approaches in sinus lift follow-up should be considered to elucidate the clinical potential of these two PLGA-based materials in dentistry.

Ethyl Glucuronide Elimination Kinetics in Fingernails and Comparison to Levels in Hair.

AIMS: Measurement of ethyl glucuronide (EtG) in nail, as a biomarker for alcohol intake, has recently been suggested as alternative to measurement in hair. The aim of this study was to compare levels of EtG in nail and hair, and to investigate the elimination kinetics of EtG in fingernails during an alcohol abstinent period. METHODS: Overall, 40 subjects (median estimated daily intake of ethanol (EDI) 92.5 g/day) were recruited from an alcohol rehabilitation clinic. Nail and hair samples were collected at inclusion and nail clippings were collected every 7-10th day for up to 12 weeks. RESULTS: All patients showed higher nail EtG/EDI ratios compared to hair EtG/EDI ratios (P < 0.001). The median value of the ratios between EtG in nail and EtG in hair was 5.0 (range: 1.07-56.1). There was a significant correlation between nail EtG/EDI and hair EtG/EDI (Spearman's ρ = 0.638, P < 0.001). EtG disappeared from nails after ~2 months of abstinence and the median calculated EtG half-life in nail clippings was 13.3 days (range: 5.5-29.0). There was a significant correlation between the time elapsed to last positive sample for nail EtG and nail EtG levels at time of inclusion (Spearman's ρ = 0.449, P = 0.004). CONCLUSION: The present data indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking. EtG may disappear faster from nail than expected from nail growth physiology. SHORT SUMMARY: Nails are an alternative matrix to hair when measuring ethyl glucuronide (EtG). The present study indicate that EtG cut-off levels in nails should be higher compared to the established 30 pg/mg EtG cut-off in hair representing heavy drinking, and EtG may disappear faster from nail than expected.