Motorneurone differenziert aus embryonalen Stammzellen bilden neuromuskuläre Endplatten In Vitro und verbessern die funktionelle motorische Regeneration In Vivo

Indroduction: We hypothesize that transplantation of embryonic stem cell derived motor neurons may enhance outcomes by better supporting the biological integrity of the injured muscle and nerve. These motor neurons may provide trophic support to the muscle by forming neo-neuromuscular junctions and up-regulating specific growth factors, preserving motor unit integrity. In the current study, we examine the functional properties of embryonic stem cell derived motor neurons in vitro, and the effect of this transplant in vivo on the functional outcome after nerve repair. Methods: Murine GFP/HB9 embryonic stem cells were differentiated into motor neurons using Retinoic Acid and Sonic Hedgehog for four days. Murine C2C12 skeletal myoblasts were plated on laminin coated dishes and differentiated to form myotubes. After formation of myotubes, co-cultures were performed with motor neurons. The formation of neuromuscular junctions was confirmed by the expression of synaptic markers using immunocytochemistry on the myotubes. In the in vivo experiment, a tibial nerve transection was performed without nerve repair, and motor neurons were immediately transplanted into the nude mouse gastrocnemius muscle or 3 weeks after denervation. Quantitative and histological analysis of gastrocnemius muscle was done at days 7 and 21 after transplantation. Additional experimental groups, in which the tibial nerve underwent repair after transplantation, were also performed. The effect of the transplants on functional recovery following nerve repair was investigated with walking track analysis in those groups. Results: GFP/HB9 embryonic stem cells were differentiated into GFP+fluorescent motor neurons. Co-cultures of motor neurons and myotubes formed neuromuscular junctions, confirmed by the expression of presynaptic markers, visicle-associated membrane protein 2 and vesicular acetylcholine transporter antibodies, and postsynaptic marker, α-bungarotoxin. In the experiment of tibial nerve transection without nerve repair, the gastrocnemius muscle injected with motor neurons was less atrophied than control PBS injected muscle at both days 7 and 21, while the muscles injected 3 weeks after denervation were not preserved. The functional recovery after nerve repair with motor neuron transplantation was evaluated with walking track analysis. It was significantly enhanced compared to PBS injected group. Conclusion: The present study confirmed the formation of neuromuscular junctions using embryonic stem cells differentiated into motor neurons in vitro. Transplantation of motor neurons prevented muscle atrophy following denervation but was not capable of rescuing denervated muscle once atrophy had occurred. Following tibial nerve repair, motor neuron transplantation improved motor functional recovery.