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

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 Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation.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.

Cyclin-dependent kinase-5 prevents neuronal apoptosis through ERK-mediated upregulation of Bcl-2

“…Approximately, 15 and 45% of cell death were observed in the differentiated cells 24 and 48 h, respectively, after the cells were left in BDNF-free medium, keeping in line with the previous study. 21 These results indicate that the differentiated SH-SY5Y-N cells depend on BDNF for survival.…”

“…21 Treatment of SH-SY5Y cells with 10 mM RA in the culture medium containing 10% fetal bovine serum resulted in neurite outgrowth that appeared on day 3 and extended on days 4 and 5 ( Figure 1a). The cells were then cultured in serum-free culture medium with 100 ng/ml BDNF for an additional 5 days.…”

“…21 SH-SY5Y-N cells remained differentiated in the BDNF-containing medium for at the least 3 weeks. In contrast, however, withdrawal of BDNF resulted in a rapid cell death of the differentiated SH-SY5Y-N cells.…”

“…19 Deprivation of neurotrophins activates mitochondrial apoptotic pathway. 20,21 BDNF has been reported to induce neuronal differentiation of human SH-SY5Y cells through activation of p35/Cdk5 kinase activity. 21 Here, we show that BDNF-induced p35/ Cdk5 kinase activity upregulates Bcl-2 through the ERK pathway during neuronal differentiation, and thus protects the differentiated neuron-like cells from DNA damage-induced apoptosis.…”

“…20,21 BDNF has been reported to induce neuronal differentiation of human SH-SY5Y cells through activation of p35/Cdk5 kinase activity. 21 Here, we show that BDNF-induced p35/ Cdk5 kinase activity upregulates Bcl-2 through the ERK pathway during neuronal differentiation, and thus protects the differentiated neuron-like cells from DNA damage-induced apoptosis. Inhibition of Cdk5 with small interfering RNA (siRNA) inhibits ERK pathway and downregulates Bcl-2 in human neurons, thus rendering them vulnerable to DNA damage.…”