Journal: Frontiers in Cell and Developmental Biology
Article Title: Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model
doi: 10.3389/fcell.2024.1429759
Figure Lengend Snippet: Evolution of marker protein expression along terminal myogenesis indicates differentiation of skeletal myotubes from hiPSC . hiPSC were determined and further differentiated for up to 60 days. (A-A′) Upper panels: schematic protocol timeline for hiPSC determination towards skeletal myogenic fate (A) and hiPSC-derived myoblast terminal differentiation (A′) . Lower panels: representative brightfield images of cell populations at various determination and differentiation timepoints. Presomitic mesoderm cells, premyogenic progenitors and mixed cell population consisting of myoblasts and myotubes were imaged at d-45, d-40 and d-22, respectively. hiPSC-derived myoblasts were obtained after 50 days of hiPSC determination (A) and were further induced to terminally differentiate into myotubes after 6 days of proliferation and 4 days of differentiation in N2-based medium (A′) . Scale bars, 100 µm. (B–F) hiPSC-derived myoblasts were differentiated according to the protocol shown in A-A′ , fixed at different timepoints, and immunostained for myogenic markers. Skeletal muscle cells were derived from control and SOD1 D90A mutant hiPSC lines. (B) Representative confocal images of samples immunostained for myogenin (MyoG) and myosin heavy chain (MYH1) at timepoints as indicated. Scale bars, 100 µm. (C,D) Quantification of the percentage of MyoG positive nuclei (C) and of percentage of nuclei within MYH1 positive cells (differentiation index; D) as a function of differentiation time. Graphs depict mean ± SD. (E,F) Quantification of MyoG (E) and MYH1 (F) mean fluorescence intensity of positive cells. At least three biological replicates were analyzed per condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Article Snippet: Myoblasts were purified and subcultured myoblasts were expanded in skeletal muscle cell growth medium (SMCGM; PELOBiotech; cat. no: PB-MH-272-0090) and further cryopreserved in SMCGM supplemented with 10% dimethyl sulfoxide and 10 μM ROCKi.
Techniques: Marker, Expressing, Derivative Assay, Control, Mutagenesis, Fluorescence
