Co-Culture of ES Cell-Derived Motor Neurons and Myotubes Show the Formation of Neuromuscular Junctions and Changes in Gene Expression

Irreversible muscle atrophy is a serious sequela of peripheral nerve injury, often resulting in disappointing results after microsurgical repair of a lesion. Previous work in the authors' laboratory has shown that embryonic stem (ES) cell-derived motor neurons (MNs) can prevent muscle atrophy by the formation of neuromuscular junctions that provide trophic support to the muscle following denervation in vivo. The main aim in the current study was to use an in vitro co-culture model of ES cell-derived MNs and myotubes to analyze changes in gene expression during the formation of neuromuscular junctions (NMJs).

Motor neurons were differentiated from murine eGFP/Hb9 embryonic stem cells using sonic hedgehog and retinoic acid for 4 days. On becoming motor neurons, the cells express green fluorescent protein (GFP). FACS analysis is used to determine the percentage of differentiation into motor neurons. At the same time, murine C1C12 skeletal myoblasts were plated in six well plates and allowed to form myotubes under the presence of DMEM media supplemented with 10% horse serum. After formation of myotubes, the GFP motor neurons were plated on top of the myotubes and cultured for 2 days. The cells were stained using the post synaptical marker, rhodamine-alpha bungarotoxin, and viewed under a fluorescence microscope. In a second setup, MNs were plated inside millipore inserts on top of a myotube layer to prevent cell-to-cell contact but allowing for the transmission of soluble signals. RNA was isolated from the MNs at 24 hr and a microarray analysis was performed at the authors' DNA core facility.

FACS analysis showed 80–85% GFP-MNs in the sample after each differentiation. Control myotubes with no motor neurons did not show staining for alpha-bungarotoxin. However, after 2 days of co-culture with GFP motor neurons, alpha-bungarotoxin was abundantly present at the locations of the motor neurons. This strongly suggests the formation of neuromuscular junctions between motor neurons and myotubes. Data from other time points were expected. Microarray analysis comparing direct co-culture and formation of NMJs and co-culture separated by a semipermeable barrier were compared. Data on the specific genes affected were forthcoming.

ES cell-derived motor neurons are capable of neuromuscular junction formation with myotubes after 2 days of in vitro co-culture. Microarray analysis will provide insight into the genes involved in this process and potentially allow a more enhanced or prolonged trophic effect on denervated muscle in vitro by the transplantation of MNs.