Extracellular vesicles (EVs) are secreted by cells into the extracellular space, EVs have therapeutic potential as EVs possess neuroprotective properties. EVs are gaining attention as a new method used for neuroregenerative therapies without the need for neurosurgical intervention. Astrocytes provide a good feeder layer which is known to release growth factors into the culture media that support the growth of target cells. Astrocyte feeder layers provide extracellular secretions to help another cell proliferate. My hypothesis is that astrocytes extracellular secretions into the culture media will increase neurite length of glutamatergic neurons. Cells obtained from patient skin punch biopsies are differentiated into fibroblasts and then differentiated to induced pluripotent stem cells (iPSCs). Neural stem cells (NSCs) derived from iPSCs were differentiated into induced glutamatergic neurons with a 14-day protocol. NSCs underwent transduction with ngn2 and rtTA 1 day after seeding onto 96-well plate. Doxycycline was used to induce the overexpression of ngn2 and rtTA until days in vitro (DIV) 9. Transduced cells were selected with puromycin on DIV 2. Cells were cultured in neural expansion media DIV -2 to 0 with EGF and FGF growth factors, DIV 1 to DIV 14 in neural maturation media with BDNF and GDNF growth factors. Astrocytes were cultured and matured with a 42-day protocol. Astrocyte culture media was removed when astrocytes matured. Astrocyte differentiation media that had not touched cells was also obtained. Both medias went through centrifugation in 3 kDa and 100 kDa centrifugal filter tubes, at 4,000 g and 15ºC for 10 minutes. Concentrated medias were placed on mature glutamatergic neurons at concentrations of 10%, 20%, 30%, 40%, and 50%. Neurite length was measured with Incucyte, a quantitative live cell analysis system. Image captured at hour 0 prior to placing concentrated medias. Serial images captured at 24, 48, 72-hour intervals after concentrated medias were placed. Glutamatergic neuronal networks did not appear to be in abundance or healthy, producing inconsistent results. In conclusion, the majority of glutamatergic neurons did not form the amount of distinct neuronal networks necessary for accurate Incucyte analysis of neurite length.
