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.
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 Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation.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.
Grup de Neurobiologia Molecular, Department de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Lleida, Catalonia, Spain.
M
M
Encinas
M
M
Iglesias
Y
Y
Liu
H
H
Wang
A
A
Muhaisen
V
V
Ceña
C
C
Gallego
J X
JX
Comella
D017209:Apoptosis / Q000187:drug effects; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009447:Neuroblastoma; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016875:tau Proteins / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D008869:Microtubule-Associated Proteins; D016900:Neurofilament Proteins; D016875:tau Proteins; C051774:neurofilament protein H; D014212:Tretinoin; D020169:Caspases