Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro

Abstract

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6-to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic ␣-toward muscle-specific ␤-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides

“…C2C12 myoblasts [purchased from PHE (Public Health England) culture collections: ECACC, Porton Down, Salisbury, UK] were cultured in growth media: DMEM (Dulbecco’s Minimum Essential Medium, with high glucose, and containing Glutamax) (Gibco, Grand Island, NY, USA) with 20 % FCS (Fetal Calf Serum) (GIBCO) and 1 % penicillin/streptomycin (GIBCO) following the recommended protocol, and differentiated in DMEM supplemented with 2 % equine serum and 1 % penicillin/streptomycin. C1F cells were isolated as a clonal line from neonatal muscle, from the H2 Kb-tsA58 transgenic mouse (Morgan et al 1994 ) in the Peckham laboratory (Peltzer et al 2008 ), and cultured as described previously (Morgan et al 1994 ; Peltzer et al 2008 ). Briefly, myoblasts were proliferated at 33 °C in the presence of IFN-γ (Becton Dickinson Biosciences, San José, CA, USA) in standard growth medium (DMEM, high glucose, Glutamax, 20 % FCS, 1 % chick embryo extract (CEE) (CEE was prepared as described by Dabiri et al 1999 .…”

“…This makes it challenging to use these cells to investigate muscle differentiation, and sarcomeric organisation. We tested three different types of cultured muscle cells: the commonly used mouse C2C12 cell line (Blau et al 1983 ), a conditionally immortalised mouse muscle cell clone, C1F, developed by us from the ‘immorto’ mouse (Morgan et al 1994 ; Peltzer et al 2008 ), and primary human skeletal muscle cells that are commercially available.…”

Transitions towards either slow‐oxidative or fast‐glycolytic phenotype can be induced in the murine WTt myogenic cell line

“…In order to obtain insights into the mechanisms of coordinated regulations involved in normal or pathological skeletal muscle differentiation processes, we have chosen to develop new murine cell lines able to differentiate into myofibers expressing adult contractile markers in culture conditions. Such a cellular model is lacking as rodent muscle cell lines or primary cultures of satellite cells do not reach differentiation stages with expression of adult contractile markers [see references in Peltzer et al, 2008]. Therefore, muscle clonal cell lines were derived from satellite cells of the transgenic H‐2kb‐tsA58 mouse also called immortomouse [Morgan et al, 1994].…”

“…Therefore, muscle clonal cell lines were derived from satellite cells of the transgenic H‐2kb‐tsA58 mouse also called immortomouse [Morgan et al, 1994]. These cells will develop a sarcomeric apparatus expressing fast and/or slow adult myosin heavy chain (MHC) isoforms in culture, contrary to commonly used rodent cell lines [Miller et al, 2003; Peltzer et al, 2008]. We have shown that clones derived from slow‐oxidative muscles will differentiate preferentially into myotubes of slow contractile phenotype when submitted to optimal culture conditions [Peltzer et al, 2008].…”

“…These cells will develop a sarcomeric apparatus expressing fast and/or slow adult myosin heavy chain (MHC) isoforms in culture, contrary to commonly used rodent cell lines [Miller et al, 2003; Peltzer et al, 2008]. We have shown that clones derived from slow‐oxidative muscles will differentiate preferentially into myotubes of slow contractile phenotype when submitted to optimal culture conditions [Peltzer et al, 2008]. One cell line derived from newborn satellite cells, named WTt clone, has been analyzed in more details, using markers previously developed for rodent in vivo studies dealing with coordinated modifications that take place in striated muscles [Hourdé et al, 2005; Noirez et al, 2006].…”