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.