In vitro characterization of proliferation and differentiation of trout satellite cells

Abstract

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named "reserve" cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.

In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous

“…Atlantic cod myosatellite cells were able to proliferate and differentiate in vitro. The morphological characteristics of Atlantic cod satellite cells were similar to the reported features found in other teleosts species, particularly their spindle-shape morphology [21,22,28,29]. The observed irregular shape of some satellite cells at the early days of culture could have been due to the enzymatic digestion and mesh passages.…”

Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing

“…Compared to cells cultured in 5% FBS, differentiation is substantially delayed in cells cultured in 1% or 2% FBS, although 2% allows cells to proliferate while 1% produces quiescence. This is in contrast to immortalized muscle cell lines as primary myosatellites from a variety of vertebrates differentiate in response to high serum concentrations (Rando & Blau 1994, Thomas et al 1994, Manceau et al 2008, Beermann et al 2010, Bower & Johnston 2010a, Gabillard et al 2010, Zhu et al 2010. In addition, rainbow trout cells spontaneously differentiate at low cell densities and high serum levels (Matschak & Stickland 1995, Castillo et al 2004, Montserrat et al 2007, Diaz et al 2009, Bower & Johnston 2010a, which also differs from immortalized cell lines.…”

“…The suspension was then sequentially filtered through 70 and 40 mm nylon cell strainers, centrifuged, resuspended, and counted before plating. Cells isolated using this procedure have 90% purity for myosatellite cells (Koumans et al 1990, Fauconneau & Paboeuf 2001, Gabillard et al 2010.…”

“…To quantify the effects of myostatin and IGF-1 on the spontaneous differentiation of proliferating myosatellite cells, which is known to occur in fish primary cells (Matschak & Stickland 1995, Rescan et al 1995, Vegusdal et al 2004, Montserrat et al 2007, Diaz et al 2009, Bower & Johnston 2010a, Gabillard et al 2010, the expression of markers for early (Myf5 and MyoD1), mid (myogenin), and late (MLC) differentiation was measured (Perry & Rudnick 2000, Rudnicki et al 2008. Differences in individual marker expression were noted, although for the most part, the patterns were similar for all markers.…”

“…These quiescent stem cells are activated in response to exercise or injury and ultimately contribute to hypertrophic growth as they differentiate and fuse to mature fibers (Grounds & Yablonka-Reuveni 1993, Pavlath & Horsley 2003, Charge & Rudnicki 2004. Some of the underlying molecular mechanisms involved appear to be fairly well conserved in all vertebrates (Buckingham & Vincent 2009) as primary myosatellite cells from a variety of species spontaneously differentiate and respond similarly to growth factors (Thomas et al 1994, Galvin et al 2003, Castillo et al 2004, Manceau et al 2008, Bower & Johnston 2010a, Gabillard et al 2010, Zhu et al 2010. Unlike birds and mammals, however, where muscle hyperplasia stops after birth, both hyperplastic and hypertrophic growth occurs in fish muscle (Mommsen 2001).…”