Discussion

From the results of this experiment, there does not appear to be very much labeling of Alpha 1C subunits on the embryonic stem cells that express GFP, which would signify the presence of voltage-gated calcium channels on these cells.  It is known that these mechanisms are required for the proper functioning of the neuron, so it is quite significant that they are not obvious in their existence on the embryonic stem cells.  If these calcium channels are not present on the differentiated cells, their electrophysiological functions are not completely present, and the cells would not be successful in their neuron functioning.  Further experiments are required to establish why these ion channels are not present. For example, the addition of certain growth factors to the culture medium or increasing the length of time the cells are co-cultured may lead to an expression of these channels.

 In the control for this experiment, NCAM clearly labeled the muscle fibres of the chick muscle.  There was no alpha-1 C labeling of the chick muscle.  Fig. 18 showed a red colour, but as there were no punctate red marks in the muscle tissue alone and therefore no clear alpha-1 C labeling; there may have been non-specific labeling of the muscle tissue.  Pictures of the embryonic stem cells showed the cells beginning to leave the cluster of cells and to extend processes outward as marked by the expression of the Green Fluorescent Protein (GFP,) with cell processes of varying sizes and lengths.  There was not much obvious Alpha 1 C labeling of the embryonic stem cells, but in Fig. 7 there seemed to have been some clustering of Alpha 1 C labeling around cell-shaped bodies.  The co-culture of chick muscle and embryonic stem cells was interesting in that there appeared to be a correlation between Alpha 1 C punctate labeling and NCAM labeling of muscle fibres.  The overlays in figures 11 and 14 showed this co-localization, and it appears as though there were Alpha 1 C subunits on the muscle fibres in the presence of embryonic stem cells that were not present in the control of muscle cells alone. 

 As mentioned in the results, there was no specific Alpha 1 C labeling in the control trials where chick muscle was grown independently.  In contrast, interesting results arose from the trials in which chick muscle was in co-culture with embryonic stem cells.  Pictures show punctate red marks signifying Alpha 1 C labeling on what appears to be the muscle fibres in Figs. 11 and 14 where there was no labeling in the muscle control.  It is postulated that the embryonic stem cells induced the creation of voltage-gated calcium channels in the chick muscle during their development in co-culture.  There is not much obvious Alpha 1 C labeling on the green embryonic stem cells, but in Fig. 7 there seems to be some clustering of Alpha 1 C labeling around cell-shaped bodies. It is possible that some cells which have not differentiated into motor neurons, i.e. they are not green, express this channel.  An experiment could be designed to identify on which cells the Alpha 1 C labeling was occurring.  After the chick muscle cells and mouse embryonic stem cells were co-cultured, they would be treated with Alpha 1 C and NCAM antibodies as in this experiment, but also with an antibody specific to all mouse cells.  Then, one would be able to establish if there is a correlation between either the Alpha 1 C labeling and mouse embryonic stem cells or between the either the Alpha 1 C labeling and chick muscle cells.   

Amyotrophic Lateral Sclerosis is a degenerative neurological diseases characterized by the spontaneous death of motor neurons, which leads to the wasting of muscle cells.  There are short-term treatments available for this and other degenerative neurological diseases, but no cures for the diseases.  Embryonic stem cells may serve as a source of neurons to repopulate deceased neurons in the body of patient with Amyotrophic Lateral Sclerosis, especially now as techniques to direct their differentiation to specific neurons of the Central Nervous System are defined.  Furthermore, embryonic stem cells may generate the molecular characteristics of a motor neuron such as extending axons out of spinal cord and forming functional synaptic contacts with target muscles, but the electrophysiological functions of these differentiated stem cells, or the capacity of these cells to function as motor neurons, must be explored for them to be used in treatments of the aforementioned diseases.  This experiment investigates the capacity of the embryonic stem cell to function as a motor neuron by looking for the presence of voltage-gated calcium channels crucial to its functioning as a motor neuron, one step toward ascertaining whether their use is feasible in treatment. 

Conclusion

There does not appear to be much labeling of Alpha 1C subunits on the embryonic stem cells that express GFP, which would signify the presence of voltage-gated calcium channels on these cells; if these calcium channels are not present on the differentiated cells, their electrophysiological functions are not completely present, and the cells would not be successful in their neuron functioning.  The presence of calcium channels in differentiated mouse embryonic stem cells has not fully be defined, and further experiments are required to establish why these ion channels are not present.

 There was no specific Alpha 1 C labeling in the control trials where chick muscle was grown independently, but there are punctate red marks signifying Alpha 1 C labeling on what appears to be the muscle fibres when chick muscle was in co-culture with embryonic stem cells where there was no labeling in the muscle control.  It is postulated that the embryonic stem cells induced the creation of voltage-gated calcium channels in the chick muscle during their development in co-culture.  This is a significant result; the differentiated stem cells were in fact communicating with the muscle cells, and this is the fundamental principle of the nervous system.  This experiment’s investigation of the capacity of embryonic stem cell to function as a motor neuron by looking for the presence of voltage-gated calcium channels shows the distinct possibility of using differentiated stem cells in treatment of Amyotrophic Lateral Sclerosis.