NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings

Abstract

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.

Skeletal muscle-derived cell implantation for the treatment of sphincter-related faecal incontinence

“…6a, c ). However, Nanog, a stem cell marker previously described to be expressed in mesenchymal stem cells [ 40 ] and functionally required for conserved myogenic differentiation potential [ 41 ], was expressed in a medium percentage of SMDC (Fig. 6 ).…”

Efficient and high yield isolation of myoblasts from skeletal muscle

“…Furthermore, the mRNA expression profile of myogenic regulatory factors suggests that the cells in culture were mostly proliferating myoblasts as evidenced by higher levels of early myogenic regulatory factors Myf5 and MyoD and lower levels of late differentiation markers, MyoG and MRF4 (Fig. 3E) (Yablonka-Reuveni, 2011; Zanou and Gailly, 2013; Shahini et al, 2018). Upon reaching confluence, the myoblasts exhibited pronounced myogenic differentiation capacity as evidenced by formation of multinucleated myotubes capable of spontaneous contraction.…”

“…Regeneration of adult skeletal muscle relies on the activation, proliferation and fusion of these myogenic progenitors into degenerated myofibers (Yablonka-Reuveni, 2011; Yin et al, 2013). Isolation of myogenic progenitors from the skeletal muscle niche provides us an in vitro test bed to study muscle physiology and enables studies on the intrinsic and extrinsic factors affecting myogenic differentiation (Shahini et al, 2018) as well as cellular and molecular pathways that can lead to muscle atrophy or dystrophy (Bareja and Billin, 2013). In vivo assessments of cellular function can also be done by delivering cells to the skeletal muscle of animal models in order to test their contribution to muscle regeneration after injury or disease.…”

Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance

“…Hsiao et al (2009) reported a DNA oligonucleotide based micropatterned glass substrate for selective adherence and subsequent differentiation of primary myoblasts. Bajaj et al (2011) Antigny, Koenig, Bernheim, & Frieden, 2013;Bettadapur et al, 2016;Shahini et al, 2018). PDMS membranes were utilised for micropatterning C2C12 cells by Huang, Lee, and Li (2010) to study myoblast alignment.…”

“…Zatti et al (2012) went a step further by micropatterning C2C12 cells on hydrogels with tissue like stiffness and elucidating the importance of secretory factors in myogenesis. Furthermore, several other studies have utilized morphometric based characterization parameters, for example, myotube width, fusion index, and nuclear distribution for assessing differentiation and myotube formation (Agley, Velloso, Lazarus, & Harridge, 2012;Antigny, Koenig, Bernheim, & Frieden, 2013;Bettadapur et al, 2016;Shahini et al, 2018). In addition, techniques like 2D FFT analysis and orientation order parameter (OOP) have been applied for assessing alignment in muscle tissues (Bajaj, Rivera, Marchwiany, Solovyeva, & Bashir, 2014;Drew, Eagleson, Baldo Jr., Parker, & Grosberg, 2015;Feinberg et al, 2012).…”