Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Abstract

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

Evolutionary history and epigenetic regulation of the three paralogous pax7 genes in rainbow trout

“…Of particular importance have been in vitro studies, where MSCs can be isolated to assess their potential contribution to the muscle growth of these fish (Gabillard et al 2010, Froehlich et al 2014). Using such an in vitro model for experimentation, Neil Bower and Ian Johnston (Bower and Johnston 2010) documented that pax7 expression is up regulated during the differentiation of MSCs from Atlantic salmon ( Salmo salar ).…”

“…For all studies, MSCs were isolated from juvenile rainbow trout ( Oncorhynchus mykiss ) (15-45 g, depending on culture and availability) essentially as previously described (Froehlich et al 2014). Isolated MSCs were plated on poly-L-lysine and laminin-coated plates at 200,000 cells per cm 2 in 6-well (chromatin immunoprecipitation, ChIP; or real-time quantitative PCR, RT-qPCR) or 24-well (immunocytochemistry) plates and cultured in 10% FBS-DMEM for 4 days (with cell fixation on days 2 and 4) and 2% FBS-DMEM for an additional 4 days (with cell fixation on day 8 post-isolation).…”

Trout myomaker contains 14 minisatellites and two sequence extensions but retains fusogenic function

“…Satellite cells from trout white muscle (5-10 g) were cultured as previously described (22,33). Briefly, after several enzymatic digestions and cell filtration steps, the cells were seeded on glass coverslips at a density of 160,000 cells/cm 2 and incubated for 40 min.…”

miR-210 expression is associated with methionine-induced differentiation of trout satellite cells

“…Satellite cells were extracted from the white muscle of trout (5-10 g), as previously reported (Froehlich et al, 2014;Gabillard et al, 2010). Briefly, after several enzymatic digestion and cell filtration steps, the cells were seeded at a density of 160,000 cells cm −2 and left for 40 min.…”