Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-b pathways-both of which were impaired in senescent cellsleading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. STEM CELLS 2017;35:207-221 SIGNIFICANCE STATEMENTAging severely hinders the properties of adult cells resulting in stem cell pool exhaustion, defective tissue regeneration and progression of age related pathologies. Therefore, there is an unmet need to reverse the effects of aging on adult cells. Here, we demonstrate that the expression of the embryonic gene, NANOG, is sufficient to reverse aging and completely restore the capacity of aged stem cells to form muscle and contract. We also illustrate a novel role of NANOG as a regulator of the myogenic machinery. Collectively, our work demonstrates that age-associated cellular dysfunction can be reversed, thereby increasing the potential of stem cells from aged donors for cellular therapies and tissue regeneration.

Bone healing can be significantly expedited by applying electrical stimuli in the injured region. Therefore, a three-dimensional (3D) ceramic conductive tissue engineering scaffold for large bone defects that can locally deliver the electrical stimuli is highly desired. In the present study, 3D conductive scaffolds were prepared by employing a biocompatible conductive polymer, ie, poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) (PEDOT:PSS), in the optimized nanocomposite of gelatin and bioactive glass. For in vitro analysis, adult human mesenchymal stem cells were seeded in the scaffolds. Material characterizations using hydrogen-1 nuclear magnetic resonance, in vitro degradation, as well as thermal and mechanical analysis showed that incorporation of PEDOT:PSS increased the physiochemical stability of the composite, resulting in improved mechanical properties and biodegradation resistance. The outcomes indicate that PEDOT:PSS and polypeptide chains have close interaction, most likely by forming salt bridges between arginine side chains and sulfonate groups. The morphology of the scaffolds and cultured human mesenchymal stem cells were observed and analyzed via scanning electron microscope, micro-computed tomography, and confocal fluorescent microscope. Increasing the concentration of the conductive polymer in the scaffold enhanced the cell viability, indicating the improved microstructure of the scaffolds or boosted electrical signaling among cells. These results show that these conductive scaffolds are not only structurally more favorable for bone tissue engineering, but also can be a step forward in combining the tissue engineering techniques with the method of enhancing the bone healing by electrical stimuli.

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Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.

Senescence of myogenic progenitors impedes skeletal muscle regeneration. Here, we show that overexpression of the transcription factor NANOG in senescent myoblasts can overcome the effects of cellular senescence and confer a youthful phenotype to senescent cells. NANOG ameliorated primary hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction. The rejuvenating effects of NANOG included restoration of DNA damage response via up-regulation of DNA repair proteins, recovery of heterochromatin marks via up-regulation of histones, and reactivation of autophagy and mitochondrial energetics via up-regulation of AMP-activated protein kinase (AMPK). Expression of NANOG in the skeletal muscle of a mouse model of premature aging restored the number of myogenic progenitors and induced formation of eMyHC + myofibers. This work demonstrates the feasibility of reversing the effects of cellular senescence in vitro and in vivo, with no need for reprogramming to the pluripotent state.

Mesenchymal stem cells (MSCs) have been extensively used in the field of tissue engineering as a source of smooth muscle cells (SMCs). However, recent studies showed deficits in the contractile function of SMCs derived from senescent MSCs and there are no available strategies to restore the contractile function that is impaired due to cellular or organismal senescence. In this study, we developed a tetracycline-regulatable system and employed micropost tissue arrays to evaluate the effects of the embryonic transcription factor, NANOG, on the contractility of senescent MSCs. Using this system, we show that expression of NANOG fortified the actin cytoskeleton and restored contractile function that was impaired in senescent MSCs. NANOG increased the expression of smooth muscle α-actin (ACTA2) as well as the contractile force generated by cells in three-dimensional microtissues. Interestingly, NANOG worked together with transforming growth factor-beta1 to further enhance the contractility of senescent microtissues. The effect of NANOG on contractile function was sustained for about 10 days after termination of its expression. Our results show that NANOG could reverse the effects of stem cell senescence and restore the myogenic differentiation potential of senescent MSCs. These findings may enable development of novel strategies to restore the function of senescent cardiovascular and other SMC-containing tissues.