Short Communication: The Effect of Different Concentrations of Methylprednisolone on Survival, Proliferation and Migration of Neural Stem/Progenitor Cells

Introduction: To address the question whether combination of methylprednisolone (MP) as an anti-inflammatory drug used in neurodegenerative diseases and neural stem/progenitor cells (NS/PCs) is safe, the present study was designed. Methods: Embryonic rat NS/PCs were exposed to different doses of MP and survival by MTT assay, proliferation by analyzing the number and diameter of neurospheres and the migration of the cells by neurosphere assay were evaluated. Results: The viability of NS/PCs reduced following exposure to 10, 15 and 20 µg/ml of MP. In addition, although the number of neurospheres didn’t change, exposure to different doses of MP resulted in formation of smaller neurospheres. Despite these undesirable effects, the highest concentration of MP (20 μg/ml) increased the migration capacity of the NS/PCs. Conclusion: The combination of MP and NS/PCs is not recommended due to the adverse effects of MP on survival and proliferation of NS/PCs.

Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice

Background: The neurosphere assay is a powerful in vitro tool to investigate neural stem cells in the dorsal lateral ventricle (dLGE). In the dLGE, metrics of sizes and numbers of neurospheres generated using this assay has not been completely characterized. The objective of this protocol is to provide a stepwise method from a single isolation that predicts the average number of neurospheres generated and to estimate an approximation of its sizes after several days in vitro. The advantage of this protocol is that no expensive and specialized equipment is needed for tissue isolation. Estimates about the numbers and sizes of neurospheres will provide investigators with quantitative data to advise on how much starting dLGE tissue is required to generate the appropriate number of spheres for the implementation of downstream applications, including immunocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro (DIV). Furthermore, we observed that this method yields an average range of neurospheres sizes greater than 50 μm, but less than 100 μm after 7 DIV. Lastly, using an anti-GFAP antibody, we show that these neurospheres can be stained, confirming their use in future immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of primary neurospheres that will be useful for further advances in the area of stem cell biology.