Optimized Protocol for Robust Differentiation of SH‐SY5Y Cells into Neuronal‐like Cells: An in vitro Model of Human Neuronal Cultures

The SH‐SY5Y cell line, derived from a human glioblastoma, grow and divide indefinitely under normal conditions, but have the potential to differentiate into neuronal‐like cells when treated with retinoic acid (RA). However, the extent of differentiation has been contested, leading to a variety of protocols to induce differentiation. We took a systematic approach to test the various conditions and treatments used in previously published studies to develop an optimized protocol that yields robust differentiation in an efficient manner. In morphological assays, robust and extensive processes from cell bodies and network development between cells in culture were established when brain‐derived neurotrophic factor (BDNF), B‐27, and potassium (K + ) were supplemented to RA, that survived up to 10 days in culture. One‐day treatment of RA and BDNF during differentiation was as effective as prolonged post‐differentiation treatment of BDNF, thus reducing the amount of BDNF required. Conversely, supplements of vitamin B12, AraC, and dB‐cAMP did not provide any significant difference in differentiation. In RT‐qPCR analyses, our optimized protocol led to significant increased expression of microtubule associated protein tau (MAPT) and microtubule associated protein 2 (MAP2), both of which are markers of neuronal cells. We are validating the sub‐cellular localization of these proteins via immunohistochemistry. This optimized protocol thus differentiates SH‐SY5Y cells into robust and reliable neuronal‐like cells, which can be used as a model of human neuronal cells and neurodevelopment in a reproducible in vitro system, without the ethical concerns or practical limitations of using fetal cells for human neuronal cultures. Support or Funding Information Ohio Wesleyan University: Department of Botany and Microbiology, Neuroscience Program, Summer Science Research Program, and Theory‐to‐Practice Grant.