Neuregulin

The procedure for isolation of Schwann cells from neonatal rats was approved by the Institutional Animal Care and Use Committee (IACUC) and completed at Wichita State University in Wichita, KS. Rat Schwann cells were isolated from the sciatic nerves of neonatal rats (postnatal day P1–3 rats), as described previously (Brockes et al., 1979 (link); Chen et al., 2011 (link)). After the nerves were dissected from the neonates, connective tissue was separated and removed from the nerves. Then the nerves were cut into small pieces and digested with Hank's Balanced Salt Solution containing trypsin and collagenase. The dissociated cells were collected for culture by centrifugation. For routine culture, rat Schwann cells were cultured in cell culture dishes coated with Poly-D,L-ornithine (5 mg/ml in PBS, Sigma-Aldrich, St. Louis, MO). The cells were fed with Dulbecco's Modified Eagle's Medium (Lifetechnology, Grand Island, NY) with 10% fetal bovine serum supplemented with 10 ng/ml neuregulin (R&D Systems, Minneapolis, MN) and 5 μM Forskolin (Sigma-Aldrich, St. Louis, MO) and cultured in a 37°C incubator with 5 % CO2. Cells between passages 2 and 4 were used in all experiments.

Muscle implants were fabricated as previously described^{11 (link)}. Briefly, MPC’s from F344 rats were suspended in type I oligomeric collagen (2.0 mg/mL) and flowed through a 4mm cylindrical mold. Once polymerized, implants were cultured under passive tension in Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific, Chicago, IL) supplemented with 1% penicillin, streptomycin, amphotericin B (PSF-1; HyClone, Logan, UT), and 10% fetal bovine serum (HyClone; Logan Utah) at 37^oC and 5% CO₂ for 5 days with medium changes every 2 days. On day 5, medium was changed to differentiation medium, representing DMEM supplemented with 8% horse serum (HyClone) and 1% PSF-1, and constructs were cultured for an additional 7 days to induce myotube formation.
For a subset of experiments, MPCs were induced to express motor endplates (MEE) as described previously^{14 (link)}. Briefly, muscle constructs were allowed to differentiate for 5 days at which point acetylcholine chloride (40 nM; Tocris Bioscience, Bristol, England), agrin (10 nM; R&D Systems, Minneapolis, Minnesota), and neuregulin (2 nM; R&D Systems) were added to the medium. Constructs were cultured an additional 7 days with medium changes every 3 days. Motor endplate expression was confirmed by immunostaining with Alexa Fluor 594 conjugated α-bungarotoxin (Molecular Probes, Eugene, Oregon).