Evaluating the function of motoneuron-like cells differentiated from rat adipose derived stem cells through calcium ion imaging and investigating the synaptic vesicle recycling

Cell replacement therapy has provided the basis for future clinical applications to treat neurodegenerative diseases، such as motoneuron diseases. Induced functional motoneurons are an option for replacing the lost motoneurons. Adipose derived stem cells (ADSCs) are an appropriate source of cells for autologous cell therapy with the ability of neural differentiation. In this study we use calcium ion imaging and synaptic vesicle release in order to indicate the functionality of motoneurons from adipose derived stem cells. In the present study، ADSCs were induced to motoneuron-like cells (MNLCs) by SHH and retinoic acid (RA) as inducers. ADSCs markers such as CD90، CD 49d، CD106، CD45، were measured by immunocytochemistry analysis and their multipotency were evaluated by incubation of the ADSCs with adipogenic، chondrogenic، osteogenic induction media. In addition to RT-PCR، immunocytochemistry was utilized in order to approve the expression of islet-1، oligo-2 and HLXB9 in induced MNLCs from ADSCs. To identify the functional MNLCs، a co-culture preparation of MNLCs and myocytes، calcium ion imaging and synaptic vesicle release was used. ADSCs treated with a mixture of preinducer (B27، EGF، and bFGF) and inducers factors (SHH and RA) adopted a morphology similar to motoneuron cells. Immunocytochemical staining and RT-PCR approved the treated cells expressed the motoneuron markers islet-1، oligo-2 and HLXB9. The co-cultured with myocytes indicates the formation of neuromuscular connections between MNLCs and myocytes. After two week، MNLCs showed high HLXB9 expression، indicative of full differentiation. Also، the release rate of synaptic vesicles using FM1-43 in the induced MNLCs was approved. Moreover، calcium imaging with fluo-2 results approved that functional excitatory synaptic connections can influence the activity of MNLCs. These results indicate ADSCs can be differentiated to a functional MNLCs phenotype and may be of benefit for treatment of motoneuron diseases.