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1. Preparation of extracellular matrix (ECM)-coated plates
- Dilute ECM (see Table of Materials) with ice cold 1x PBS to a final concentration of 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.
- Place 1 coverslip into each well of a 6-well plate. Add 1.5 mL of 2% ECM into each well.
- Incubate the well plate with 2% ECM for 2 h at 37 °C.
- Aspirate ECM from the well plate and store the plate at 4 °C before use.
2. Differentiation of iPSCs toward NMJ
- 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 201B7MYOD iPS cell line was used.
- Remove the medium from the iPSCs on the 6 cm culture dish and wash the iPSCs once with 1x PBS.
- Add 1 mL of cell detachment solution to the dish and incubate for 10 min at 37 °C.
- Add 3 mL of primate embryonic stem (ES) cell medium to the dish and gently pipette 3 times.
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- Observe the differentiated NMJ by phase inverted microscopy on day 30. The NMJ can be used for the following analysis.