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

Wash the cells with 3 milliliters of PBS before adding 1 milliliter of cell detachment solution to a Petri dish. After 10 minutes at 37 degrees Celsius, add 3 milliliters of primate embryonic stem cell medium to each well, and gently pipette three times to dissociate the cells from the coverslips.

Next, pool the detached stem cell containing supernatants into a 50-milliliter conical tube for centrifugation, and resuspend the stem cell pellet in 3 milliliters of fresh primate embryonic stem cell medium supplemented with 10 micromolar Y27632 ROCK inhibitor for counting.

Then, dilute the cells to a 2 x 10⁵ cells per milliliter of medium, supplemented with ROCK inhibitor concentration, and return 2 milliliters of cells to each coverslip in each well of the extracellular matrix-coated 6-well plate.

After 24 hours, replace the supernatant in each well with 2 milliliters of fresh primate embryonic stem cell medium supplemented with 1 microgram per milliliter of doxycycline, and return the plate to the cell culture incubator for 24 hours.

At the end of the incubation, replace the supernatants with 2 milliliters of differentiation medium supplemented with doxycycline per well, and return the plate to the cell culture incubator for 10 days.

At the end of the incubation, replace the supernatants with 2 milliliters of myogenic differentiation medium supplemented with doxycycline per well, and return the plate to the cell culture incubator for an additional 10 days. At the end of the incubation, replace the supernatants with 2 milliliters of neuromuscular junction medium per well, and return the plate to the cell culture incubator for 30 days.