ABSTRACT
The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5(+), Pax3/Pax7(+) cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.
Subject(s)
Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism , Animals , Biomarkers , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Lineage , Cell Proliferation , Gene Expression Regulation, Developmental , Mice , Mice, Transgenic , Muscle Development , Muscle, Skeletal/physiology , PAX3 Transcription Factor/genetics , PAX7 Transcription Factor/genetics , Phenotype , Regeneration , Satellite Cells, Skeletal Muscle/transplantationABSTRACT
Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5-7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na(+)- and Ca(2+)-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca(2+) transients; fiber contractions coupled to these Ca(2+) transients suggest that Ca(2+) entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca(2+) transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration.
Subject(s)
Dermis/cytology , Lipids/chemistry , Models, Biological , Muscles/pathology , Muscles/physiopathology , Tissue Engineering/methods , Acetylcholine/pharmacology , Animals , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Gene Expression Regulation/drug effects , Ion Channel Gating/drug effects , Mice , Muscle Development/drug effects , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Muscle Fibers, Skeletal/ultrastructure , Muscles/ultrastructure , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolismABSTRACT
Hypoxic-ischemic (HI) insult during the perinatal period remains as one of the most common causes of brain injury and produces long-term neurological deficits, and there is a growing need for effective therapies. The aim of the present work was to perform a prospective study designed to assess the possible protector effect of two endocannabinoids: 2-arachidonoylglycerol (2AG) and anandamide (AEA) in the brain after HI injury in perinatal rat model. We evaluate their effects on cell death and check several cellular parameters. 7-days-old Wistar rats were assigned to four different experimental groups (n=7-10): Sham, HI, and HI treated with 2AG or AEA. The injury was induced by the left carotid artery ligature and subsequent exposure to 8% O(2) for 120 min. Immediately after the injury, treated groups received a single dose of 2AG (1mg/kg) or AEA (5mg/kg) and then animals were sacrificed 24, 72 h or 7 days after the HI event. Brains fixed by perfusion were stained with Nissl for morphological studies, and non-fixed brains were dissociated and analyzed by flow cytometry to quantify apoptosis, mitochondrial state, intracellular calcium and reactive oxygen species. Our results show that both 2AG and AEA have beneficial effects after HI injury in this rat model, producing a remarkable amelioration of brain injury, reducing apoptotic cell death, contributing to the maintenance of mitochondrial functionality, and improving cellular parameters such as the influx of calcium and ROS production.
