Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts

Abstract

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24 h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP /DsRed hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products.

Identification of the myogenetic oligodeoxynucleotides (myoDNs) that promote differentiation of skeletal muscle myoblasts by targeting nucleolin

“…Murine myoblasts (mMBs) were isolated from the skeletal muscle of 4-week-old C57BL/6J mice (Clea Japan, Tokyo, Japan) and primary-cultured as previously described (Takaya et al, 2017; Nihashi et al, 2019b). mMBs were maintained on the dishes or plates coated with collagen type I-C (Cellmatrix; Nitta Gelatin, Osaka, Japan), and cultured in growth medium (GM) for mMB consisting of Ham’s F10 medium (Thermo Fisher Scientific, MA, USA), 20% fetal bovine serum (FBS) (HyClone; GE Healthcare, UT, USA), 2 ng/ml recombinant human basic fibroblast growth factor (Wako), and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (PS) (Nacalai).…”

“…Immunocytochemistry of myoblasts was performed as previously described (Takaya et al, 2017; Nihashi et al, 2019a; Nihashi et al, 2019b). The myoblasts were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with 0.5 μg/ml mouse monoclonal anti-myosin heavy chain (MHC) antibody (MF20; R&D Systems, MN, USA) and 1.0 μg/ml rabbit polyclonal anti-nucleolin antibody (ab22758; Abcam, Cambridge, UK).…”

“…Immunocytochemistry of myoblasts was performed as previously described (Takaya et al, 2017;Nihashi et al, 2019a;Nihashi et al, 2019b).…”

Distinct cell proliferation, myogenic differentiation, and gene expression in skeletal muscle myoblasts of layer and broiler chickens

“…Myoblasts from hindlimb muscles of WL or UKC chicken embryos at HH36 (E10) were isolated, primary-cultured, and induced to differentiate as previously described 57,58 . Conventional WL and UKC chicken eggs were provided by National Federation of Agricultural Cooperative Associations (Tokyo, Japan) and were conventionally cultured at 38.5 °C and 60% humidity with 45° horizontal rotation every 3.75 min in an incubation chamber for 10 days.…”

Toll-like receptor ligand-dependent inflammatory responses in chick skeletal muscle myoblasts

“…A better understanding of TLRs in chick myoblasts may provide interesting insights into skeletal muscle tissue inflammation. Skeletal muscle myoblasts were isolated from the leg muscles of E10 chick embryos and two-day-old chickens as previously described (Takaya et al, 2017). The myoblasts were cultured on dishes coated with collagen type I-C (Cellmatrix; Nitta Gelatin, Osaka, Japan) at 37°C with 5% CO2 throughout the experiment.…”