Synthetic mammalian neuromuscular junction and method of screening for a candidate drug thereon
A method for forming neuromuscular junctions includes forming functional neuromuscular junctions between motoneurons and muscle cells by co-culturing one or more human motoneurons and one or more human muscle cells in a substantially serum-free medium. A synthetic mammalian neuromuscular junction includes a human motoneuron functionally linked to a human muscle cell in a substantially serum-free medium. An artificial substrate may be used to support the one or more neuromuscular junctions.
1. A method of screening for a candidate therapeutic drug for a neuromuscular disease, the method comprising:
co-culturing differentiated human skeletal muscle stem cells adhered to an artificial surface and overlayered with differentiated human spinal cord stem cells in a serum-free medium;
forming at least one functional neuromuscular junction between a differentiated human skeletal muscle stem cell and a differentiated human spinal cord stem cell;
contacting the at least one functional neuromuscular junction with a candidate drug;
and monitoring the at least one functional neuromuscular junction following contact with the candidate drug, wherein the monitoring comprises recording one or more electrophysiological properties of the co-culture or video recording of the co-culture.
2. The method of claim 1 , further comprising comparing the one or more electrophysiological properties of the co-culture after the contacting step to the same one or more electrophysiological properties of the co-culture before the contacting step.
3. The method of claim 2 , wherein a change in the one or more electrophysiological properties of the co-culture indicates a candidate drug.
4. The method of claim 3 , wherein the candidate drug affects neuromuscular junction synaptogenesis.
5. The method of claim 3 , wherein the candidate drug affects neuronal initiation of muscle contraction.
6. The method of claim 1 , wherein recording one or more electrophysiological properties comprises a current-clamp technique, a voltage-clamp technique, or both.
7. The method of claim 1 , wherein the serum-free medium comprises the components in Table 1.
8. The method of claim 1 , wherein the serum-free medium comprises at least one synaptogenesis promoting component and one or more trophic factors.
9. The method of claim 1 , wherein the serum-free medium comprises NbActiv4.
10. The method of claim 1 , wherein the artificial surface comprises a silicon based substrate monolayer deposited thereon.
11. The method of claim 10 , wherein the silicon based substrate monolayer comprises DETA (trimethoxysilylpropyldiethylenetri-amine).
12. The method of claim 10 , wherein the silicon based substrate monolayer is deposited on the artificial surface in a predetermined pattern.
13. The method of claim 1 , wherein the method is a high-throughput screening method.