Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derivedneurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells

A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Seque ntial exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotro phic factor in serum-free medium yields homogeneous populations of cells wi th neuronal morphology, avoiding the presence of other neural crest derivat ives that would normally arise from those cells. Cells are withdrawn from t he cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastom a hypophosphorylation, Cell survival is dependent on the continuous presenc e of brain-derived neurotrophic factor, and removal of this neurotrophin ca uses apoptotic cell death accompanied by an attempt to reenter the cell cyc le. Differentiated cells express neuronal markers, including neurofilaments , neuron-specific enolase, and growth-associated protein-43 as well as neur onal polarity markers such as tau and microtubule-associated protein 2, Mor eover, differentiated cultures do not contain glial cells, as could be evid enced after the negative staining for glial fibrillary acidic protein. In c onclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristic s of primary cultures of neurons. This model may be useful to perform large -scale biochemical and molecular studies due to its susceptibility to genet ic manipulation and the availability of an unlimited amount of cells.