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Systematic evaluation of morphological and molecular changes in SH-SY5Y cells induced by common differentiation protocols with a particular focus on the abundance of neuronal subtypes

ABSTRACT: The SH-SY5Y cell line is a triple-cloned subline of SK-N-SH cells that were originally isolated in the early 1970s from a bone marrow biopsy of a four-year-old female patient suffering from neuroblastoma. Since then, this cell line has been used as one of the major cell culture models in neuroscience and to study neurodegeneration, as it comprises many of the biochemical and functional properties of neuronal cells. Differentiation of neuronal precursor cells into a more mature phenotype represents one of the key steps and directed differentiation utilising various reagents is thought to provoke a defined neuronal subtype. Unfortunately, until now there is no consensus, which differentiation protocol shall be utilised to reach a specific neuronal subtype. Thus, the aim of the present work was to evaluate four commonly followed standard protocols for the differentiation of SH-SY5Y cells and to investigate the respective influences of varying parameters of these differentiation strategies. For this purpose, morphological analyses, mass spectrometry-based identification and quantification of specific marker proteins, time course protein expression profiling and global proteomic characterisation were conducted. On the level of morphology a low serum concentration favoured the abundance of mature neuronal cells containing long and branched neurites. Further low serum levels support the differentiation of neuroblastic cells favour the expression of dopaminergic marker proteins, in particular DDC, especially in a combined treatment utilising retinoic acid as differentiation agent. Our study clearly shows that an a priori characterisation of SH-SY5Y cells is indispensable to assess the abundance of neuronal subtypes and by that to ensure that the utilised differentiation approach is appropriately aligned with the specific research question.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Britta Eggers

LAB HEAD: Katrin Marcus

PROVIDER: PXD064335 | Pride | 2025-11-24

REPOSITORIES: Pride

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Systematic Analysis of SH-SY5Y Differentiation Protocols and Neuronal Subtype Abundance.

Prisacar Marina M Esser Svenja S Hausherr Maximilian M Karacora Bilhan B Vyushkova Yulia Y Eisenacher Martin M Grugel Robin R Marcus Katrin K Eggers Britta B

Cellular and molecular neurobiology 20251115 1

The SH-SY5Y cell line is a triple-cloned subline of SK-N-SH cells originally isolated in the early 1970s from a bone marrow biopsy of a four-year-old female patient suffering from neuroblastoma. Since then, this cell line has been used as one of the major cell culture models in neuroscience and to study neurodegeneration, as it comprises many of the biochemical and functional properties of neural precursor cells. Differentiation of neuronal precursor cells into a more mature phenotype represents ...[more]

PMID: 41239137

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Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

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