In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

This human neuromuscular junction induction system can be used to induce the formation of pre and post-synaptic components, including motor neurons, skeletal muscle, and Schwann cells. Not only can a mature, complex NMJ structure be obtained in a single culture dish from a single starting cell population, but the resulting NMJ also possesses the ability to contract. This method has potential for studying neuromuscular diseases, such as spinal muscular atrophy, and for therapeutic compound screening.

The differentiated NMJ is a very thick, complex tissue, and it's difficult to visualize. By demonstrating the procedure, we can instruct readers how to identify specific structures. Wash the cells with three milliliters of PBS before adding one milliliter of cell detachment solution to a Petri dish.

After 10 minutes at 37 degrees Celsius, add three 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 three milliliters of fresh primate embryonic stem cell medium supplemented with 10 micromolar Y-27632 ROCK inhibitor for counting. Then dilute the cells to a two times 10 to the five cells per milliliter of medium supplemented with ROCK inhibitor concentration and return two milliliters of cells to each coverslip in each well of the extracellular matrix-coated six-well plate.

After 24 hours, replace the supernatant in each well with two milliliters of fresh primate embryonic stem cell medium supplemented with one 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 two 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 two 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 two milliliters of neuromuscular junction medium per well and return the plate to the cell culture incubator for 30 days. At the end of the incubation, visualize the differentiated neuromuscular junctions by inverted microscopy. To trigger the myotube contractions, add 25 millimolar calcium chloride to each well of differentiated neuromuscular junction cells.

And within one to two minutes of treatment, place the plate on the stage of an inverted microscope. Using live cell microscopy, record a movie of the myotube contraction for 20 seconds. At the end of the recording, add 300 nanograms per milliliter of curare to the culture medium and record the movie for another 20 seconds before opening the movie file in an appropriate motion vector analysis software program for analysis.

Immunofluorescent staining of the neurofilament synaptic vessels and acetylcholine receptors can be performed to identify the neurons. Motor neurons could be identified by TUJ 1 and Islet 1 staining, while post-synaptic myotubes can be identified by staining for myosin heavy chain. Schwann cells can be labeled with S-100 antibody.

In these scanning electron microscopy images, the morphology of mature neuromuscular junctions can be observed with clear visualization of the expanded axon terminals, axons, and muscle fibers. Transmission electron microscopy can be used to visualize the mature ultra structure of the neuromuscular components, including the presynaptic axon terminal with synaptic vesicles and the post-synaptic region, which is separated by the synaptic cleft. Junctional folds mark the junction between the neuron and muscle fibers.

Here, mature axon terminals that contain synaptic vesicles are shown. Taken together, these morphological features indicate that the neuromuscular components were well-induced and matured. Functional assessment of the cells reveals that prominent neuromuscular junction motion signals can be induced by calcium chloride and that these contractions can be interrupted by curare confirming that the motor neuron signals are transmitted through the neuromuscular junction to trigger muscle contraction.

The cell seeding density influences the efficiency of the NMJ formation. It can be modified to adapt to the protocol, according to the experimental purposes. This neuromuscular junction culture contains multiple cell types, and these components can be used to study the interactions between these cells during the neuromuscular junction development or in response to specific disease pathologies.