PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

Ross Ferguson,Vasanta Subramanian

doi:10.1371/journal.pone.0159051

Abstract

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

Highlights

Investigation into fundamental mechanisms of neuroscience or therapeutic interventions can be rapidly and reproducibly performed using in vitro models such as immortalised, handled established neuroblastoma cell lines
Retinoic acid (RA) was used to induce the differentiation of SH-SY5Y and dibutyryl cyclic AMP for VSC4.1 SH-SY5Y cells were plated on coverslips coated with 0.1% gelatin in 24 well plates in DMEM:F12 (1:1) containing Glutamax supplemented with 1% Foetal bovine serum (FBS), 1% Non-essential amino acids (NEAA) and 10μM retinoic acid (RA) at a final of 5000 cells each
Undifferentiated VSC4.1 cells possess peripherin positive cell bodies with short neurites which are no longer than a single cell diameter and uniformly sized nuclei (Fig 1A-a)

Summary

Introduction

Investigation into fundamental mechanisms of neuroscience or therapeutic interventions can be rapidly and reproducibly performed using in vitro models such as immortalised, handled established neuroblastoma cell lines With this comes the problem of cell line identity as neuroblastoma cells which are typically not quiescent, lack extensive neurite projections and key marker expression characteristic of mature neurons. SH-SY5Y, a subclone of the human neuroblastoma cell line SK-N-SH derived from a metastatic bone tumour, typically shows a proliferative neuroblast-like phenotype [4] and is used extensively as a model for neurodegeneration studies Since it expresses moderate levels of dopaminergic markers such as tyrosine hydroxylase and dopamine-β hydroxylase [5] it has been a popular dopaminergic model for many years, their dopaminergic identity and suitability have been questioned [6,7]. VSC4.1 is motor neuron hybrid cell line created through

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Conclusion