In Vitro Induction of Neuromuscular Junctions from Engineered Human-Induced Pluripotent Stem Cells

1. Preparation of extracellular matrix (ECM)-coated plates

1. Dilute ECM (see Table of Materials) with ice cold 1x PBS to a final concentration of 2%
2. Add 1 mL of ECM to 49 mL of 1x PBS in a 50 mL plastic tube. Mix well by pipetting up and down several times.
3. Place 1 coverslip into each well of a 6-well plate. Add 1.5 mL of 2% ECM into each well.
4. Incubate the well plate with 2% ECM for 2 h at 37 °C.
5. Aspirate ECM from the well plate and store the plate at 4 °C before use.

2. Differentiation of iPSCs toward NMJ

1. Seed 4 x 10⁵ of iPSCs per well on the 6-well plate prepared in section 1. Make sure to seed the cells on the coverslip pre-deposited in the well.
   NOTE: In this study, the 201B7^MYOD iPS cell line was used.
   1. Remove the medium from the iPSCs on the 6 cm culture dish and wash the iPSCs once with 1x PBS.
   2. Add 1 mL of cell detachment solution to the dish and incubate for 10 min at 37 °C.
   3. Add 3 mL of primate embryonic stem (ES) cell medium to the dish and gently pipette 3 times.
   4. Collect the supernatant, which contains detached iPSCs, into a 50 mL plastic tube and centrifuge at 160 x g at 4 °C for 5 min.
   5. Carefully aspirate the supernatant, resuspend the iPSCs in 3 mL primate ES cell medium with 10 µM Y27632, and count the cell number using a hemocytometer.
   6. Dilute the iPSCs with primate ES cell medium and 10 µM Y27632 to a concentration of 2 x 10⁵ cells/mL. Add 2 mL of iPSCs on the coverslip pre-deposited in the well described in section 1.

3. Induction of the iPSCs to NMJ

1. On day 1, 24 h after seeding the iPSCs to the 6-well plate, remove the culture medium and replace it with 2 mL of fresh primate ES cell medium containing 1 µg/mL doxycycline to each well.
2. Change the medium with 2 mL of myogenic differentiation medium (MDM) (Table 1) containing 1 µg/mL doxycycline (final concentration) to each well. Refresh the medium every day from day 2 to day 10.
3. From day 11, switch the medium to 2 mL of NMJ medium (Table 1) to each well. Refresh the medium every 3‒4 days thereafter until day 30.
4. Observe the differentiated NMJ by phase inverted microscopy on day 30. The NMJ can be used for the following analysis.