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.
