Matrigel coated dishes

Satellite cell isolation and differentiation were performed as described previously with minor modifications (Hindi et al., 2017 (link)). Briefly, gastrocnemius and quadriceps muscles were excised from Lrrc8a^flfl mice (8–10 weeks old) and washed twice with 1XPBS supplemented with 1% penicillin-streptomycin and fungizone (300 µl/100 ml). Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II (2 mg/ml), 1% penicillin-streptomycin and fungizone (300 ul/100 ml) and incubated at shaker for 90 min at 37°C. Tissue was washed with 1X PBS and incubated again with DMEM-F12 media supplemented with collagenase II (1 mg/ml), dispase (0.5 mg/ml), 1% penicillin-streptomycin and fungizone (300 µl/100 ml) in a shaker for 30 min at 37°C. Subsequently, the tissue was minced and passed through a cell strainer (70 µm), and after centrifugation; satellite cells were plated on BD Matrigel‐coated dishes. Cells were stimulated to differentiate into myoblasts in DMEM‐F12, 20% fetal bovine serum (FBS), 40 ng/ml basic fibroblast growth factor (bfgf, R and D Systems, 233‐FB/CF), 1X non‐essential amino acids, 0.14 mM β‐mercaptoethanol, 1X penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM‐F12, 2% FBS, 1X insulin–transferrin–selenium, when 80% confluency was reached.

Satellite cell isolation and differentiation were performed as described previously with minor modifications [10 (link)]. Briefly, the skeletal muscles of gastrocnemius and quadriceps were excised from C57BL/6J wildtype at 8–10 weeks of age. The muscles were washed twice with 1× PBS supplemented with 1% penicillin-streptomycin and fungizone (300 μL/100 mL). DMEM-F12 media with collagenase II (2 mg/mL), 1% penicillin-streptomycin, and fungizone (300 uL/100 mL) was added to the muscles, and the muscles were shaken for 90 min at 37 °C. The tissue washing process was repeated, using DMEM-F12 media, same specifications as before except collagenase II was changed to 0.5 mg/mL, in a shaker for 30 min at 37 °C. Following this, the tissue was cut and passed through a cell strainer (70 μm). Following centrifugation, satellite cells were plated on BD Matrigel-coated dishes. Cells were differentiated into myoblasts through the usage of a mixture of DMEM-F12, 20% fetal bovine serum (FBS), 40 ng/mL basic fibroblast growth factor (bfgf, R and D Systems, 233-FB/CF), 1× non-essential amino acids, 0.14 mM β-mercaptoethanol, 1× penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/mL bfgf and then differentiated in DMEM-F12, 2% FBS, 1× insulin–transferrin–selenium, when 80% confluency was reached.

Three hiPSC lines, WT83C6 (male), CVB (male), and 4C1 (female), were derived from control individuals with informed consent and have been previously characterized elsewhere [28 (link)–30 (link)]. The hiPSC colonies were expanded on Matrigel-coated dishes (BD Biosciences, San Jose, CA, USA) with mTeSR1 medium (StemCell Technologies, Vancouver, Canada). The cells were routinely checked by karyotype and CNV arrays to avoid genomic alterations in the culture. Embryonic samples were obtained from fetal brains (10–11 weeks post-conception (PCW)) and cultured in Neurobasal (Life Technologies, Carlsbad, CA, USA) supplemented with 1X GlutaMAX (Life Technologies), 1% Gem21 NeuroPlex (Gemini Bio-Products, West Sacramento, CA, USA), 1% MEM nonessential amino acids (NEAA; Life Technologies), and 1% penicillin/streptomycin (Pen/Strep; Life Technologies). All cellular cultures were routinely tested for mycoplasma by PCR. The study was approved by the University of California San Diego IRB/ESCRO committee (protocol 141223ZF).