Background: The human neuroblastoma cell line, SH-SY5Y, has been widely used in neuroscience
research, especially in studies related to Parkinson's disease. However, differences between
clones have been demonstrated, highlighting the importance to characterize the properties of this cell
Objective: The aim of this study was to characterize the phenotype of undifferentiated and differentiated
SH-SY5Y cells using various differentiation protocols.
Methods: A morphological and quantitative analysis of markers related to dopaminergic and cholinergic
neurons, but also other phenotypes, was performed.
Results: Differentiated cells showed the typical neuronal morphology. Undifferentiated cells expressed
low levels of Tyrosine Hydroxylase (TH) and higher levels of the high-affinity Choline
Transporter (CHT1). Staurosporine (ST)-differentiation resulted in the highest number of THimmunoreactive
cells, followed by phorbol ester Phorbol-12-Myristate-13-Acetate (PMA), whereas
differentiation with Brain-Derived Neurotrophic Factor (BDNF) did not increase TH-immunoreactive
cells. TH, dopamine β-hydroxylase and vesicular monoamine transporter-2 were also significantly upregulated
in ST-differentiated cells compared to both undifferentiated and Retinoic Acid (RA)-
differentiated cells. RA induced the highest number of CHT1-immunoreactive cells while ST- and
BDNF-differentiation reduced CHT1-immunoreactive cells, indicating a decrease in the cholinergic phenotype.
The presynaptic neuronal protein, α-synuclein, was significantly upregulated in RA- and ST-treated
cells compared to undifferentiated cells. Ascorbic acid increased the number of CHT1-immunoreactive
cells in all differentiation procedures and ST-differentiated TH-positive cells significantly.
Conclusion: Our findings indicate that a quantitative characterization of the phenotype is crucial when
using SH-SY5Y cells to study the pathogenesis or evaluate compounds for treatment of neurodegenerative