Differentiated SH-SY5Y cells exhibit neuronal features but lack synaptic maturity

A vital question in neuroscience is whether and how efficiently cellular models may be differentiated into functional neuronal cells in culture. Despite the frequent use of the human neuroblastoma cell line SH-SY5Y, differentiation protocols vary extensively, with the most common being differentiation via the addition of retinoic acid and brain-derived neurotrophic factor. However, due to the lack of a reliable evaluation method, their adequacy as synaptic models remains unclear. Here, we investigate whether SH-SY5Y cells constitute a functional model for synaptic studies by phenotypically and ultrastructurally analyzing synaptogenesis in SH-SY5Y cells subjected to different differentiation protocols. Electron microscopy (EM) techniques, including conventional EM, cryo-EM, and cryo-electron tomography, were systematically applied to characterize synaptogenesis in SH-SY5Y cells. Further characterization was performed using immunostaining and functional assays, such as live exocytosis assays and whole-cell patch-clamp electrophysiology. Despite exhibiting some presynaptic-like features, differentiated SH-SY5Y cells do not form morphologically or functionally complete synapses under the conditions tested. Immunostaining results were consistent with previous findings, showing synaptic markers. However, functional investigations did not detect synaptic activity. High-throughput EM analyses revealed an absence of synaptic structures in these cells. Additionally, an alternative differentiation approach incorporating additional neurotrophic factors promoted the formation of presynaptic-like compartments containing synaptic vesicle-like vesicles (SVLVs). In contrast to typical synaptic vesicles, these SVLVs exhibited a pleomorphic size distribution and lacked connectors. These findings underscore the need for cautious interpretation of results derived from SH-SY5Y cells when investigating molecular synaptic architecture or function, as well as neurodegenerative diseases.