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

Start with engineered human iPSCs containing a myoblast differentiation or MYOD protein-encoding, doxycycline-inducible gene in a stem-cell medium containing Rho-kinase inhibitors.

Transfer the cells to ECM-coated plates and incubate to promote cell attachment.

Meanwhile, the inhibitor inactivates intracellular Rho-kinase enzymes, enhancing cell viability.

Replace the medium with a stem-cell medium containing doxycycline.

Doxycycline enters cells and binds to activator proteins. These complexes bind to the promoter, inducing the gene expression in some cells and producing MYOD proteins, which trigger stem cell differentiation into muscle cells.

Treat the cells with a myogenic differentiation medium containing myogenic differentiation factors to promote the formation of myotubes, a multinucleated muscle cell.

Later, introduce a neural induction medium with neural growth factors.

These factors differentiate the remaining iPSCs into motor neurons and Schwann cells that form a myelin sheath around the neurons.

Finally, replace the medium with a neuromuscular junction medium to promote connections between the motor neuron and myotube, forming neuromuscular junctions.

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.