Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described

Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: role of paracrine and neurohormonal milieu in the remodeling process.

BACKGROUND: In this study we investigated the effect of human amniotic fluid stem (hAFS) cells and rat adipose tissue stromal vascular fraction GFP-positive cell (rSVC-GFP) therapy and the contribution of the paracrine and neurohormonal milieu to cardiac and pulmonary vascular remodeling in a rat model of pulmonary hypertension (PH) and right heart failure (RHF). METHODS: Sprague-Dawley rats were injected with monocrotaline (MCT). Four million hAFS or rSVC-GFP cells were injected via the tail vein 3 weeks after MCT. RHF was confirmed by RV hypertrophy/dilation and by brain natriuretic peptide (BNP) level. Cytokine profile was assessed by Multiplex array. Stem cell (SC) differentiation was studied by immunofluorescence. RESULTS: MCT rats showed eccentric RV hypertrophy with increased RV dilation (measured as right ventricular mass/right ventricular volume [RVM/RVV]: MCT, 1.46 \ub1 0.12; control, 2.33 \ub1 0.24; p = 0.01), and increased RV hypertrophy (measured as LVM/RVM: MCT, 1.58 \ub1 0.06; control, 2.83 \ub1 0.1; p < 0.00001), increased BNP (MCT, 5.2 \ub1 1.2; control, 1.5 \ub1 0.1; p < 0.001) and both pro- and anti-inflammatory cytokines. SC produced a fall of BNP (hAFS, 2.1 \ub1 0.7; rSVC-GFP, 1.98 \ub1 1.3; p < 0.001) and pro-inflammatory cytokines. Positive RV remodeling with decreased RV dilation (RVM/RVV: hAFS, 1.87 \ub1 0.44; rSVC-GFP, 2.12 \ub1 0.24; p < 0.03 and p < 0.05 vs MCT) and regression of RV hypertrophy (LVM/RVM: hAFS, 2.06 \ub1 0.08; rSVC-GFP, 2.16 \ub1 0.08; p < 0.00001 vs MCT) was seen together with a decrease in medial wall thickness of pulmonary arterioles (hAFS, 35.33 \ub1 2.78%; rSVC-GFP, 37.15 \ub1 2.92%; p = 0.0001 vs MCT). CONCLUSIONS: SC engrafted in the lung, heart and skeletal muscle modulated the pro- and anti-inflammatory cytokine milieu, and produced a positive neurohormonal response. This was accompanied by positive cardiac and pulmonary vascular remodeling, with formation mainly of new vascular cells

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

textabstractSkeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, αSMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration