FIGURE 3.

NMJ functionality in 3D neuromuscular co-cultures (A) Immunocytochemical stainings of a 21 days-old neuromuscular co-culture against Tuj1, α-Actinin and DAPI. Co-cultures were generated with MNs derived from control iPSC-line #1. MNs were differentiated according to Reinhardt et al. (B) Immunocytochemical stainings of a 21 days-old neuromuscular co-culture against AChR, Tuj1 and α-Actinin. (C) Immunocytochemical stainings of a 21 days-old neuromuscular co-culture against AChR, Synaptophysin-1 and NF-H. (D) Representative calcium transient upon glutamate treatment. Each data point represents the mean of seven ROIs. Mean ± SEM. Glu, glutamate. (E) Quantification of the muscle fiber contraction. A representative curve for the muscle tissue displacement upon glutamate treatment is shown. A second curve shows the muscle displacement after pretreatment with TTX, followed by glutamate stimulation. TTX, tetrodotoxin. Glu, glutamate. (F) Quantification of the maximal muscle fiber displacement after 21 days. Two independent iPSC lines were used to generate MNs, which were subsequently used for the co-cultures. Each data point represents the maximal displacement of an individual muscle fiber tissue. Mean ± SEM. iPSCs#1: Glu, n = 17; TTX + Glu, n = 4; BoNT + Glu, n = 6. iPSCs#2: Glu, n = 15; TTX + Glu, n = 2. Muscle only, n = 13. (G) Representative low magnification images of co-cultures stained for NF-H and DAPI at the time points indicated. (H) Quantification of the axon outgrowth. Two independent iPSC lines were used to generate MNs, which were subsequently used for the co-cultures. iPSCs#1: Day 9, n = 7; Day 21, n = 4; Day 42, n = 11. iPSCs#2: Day 9, n = 7; Day 21, n = 8; Day 42, n = 10. (I) Quantification of the maximal muscle fiber displacement. Co-cultures were stimulated with glutamate and the tissue movement was analyzed at the time points indicated. iPSCs#1: Day 14, n = 13; Day 21, n = 10; Day 28, n = 16; Day 42, n = 7. iPSCs#2: Day 14, n = 5; Day 21, n = 15; Day 35, n = 6.