Lippincott Williams & Wilkins, Inc., Philadelphia
Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells
Article first published online: 4 JAN 2002
Journal of Neurochemistry
Volume 75, Issue 3, pages 991–1003, September 2000
How to Cite
Encinas, M., Iglesias, M., Liu, Y., Wang, H., Muhaisen, A., Ceña, V., Gallego, C. and Comella, J. X. (2000), Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells. Journal of Neurochemistry, 75: 991–1003. doi: 10.1046/j.1471-4159.2000.0750991.x
Abbreviations used: BNDF, brain-derived neurotrophic factor; BrdU, 5-bromo-2′-deoxyuridine; cdk, cyclin-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; GAP-43, growth-associated protein-43; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; NGF, nerve growth factor; NF-L, -M, and -H, low-, medium-, and high-molecular-weight neurofilaments, respectively; NSE, neuron-specific enolase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, PCD, programmed cell death; pRB, retinoblastoma susceptibility gene product; RA, retinoic acid; SDS, sodium dodecyl sulfate; Trk, tyrosine receptor kinase; TPA, 12-O-tetradecanoylphorbol 13-acetate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
- Issue published online: 4 JAN 2002
- Article first published online: 4 JAN 2002
- SH-SY5Y cells;
- Neurodegenerative disease;
- Cell cycle
Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2′-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.