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Neuronal Cell Lines As Model Dorsal Root Ganglion Neurons: A Research Article Molecular Pain Volume 12: 1–17 ! The Author(s) 2016 Neuronal cell lines as model Reprints and permissions: sagepub.co.uk/journalsPermissions.nav dorsal root ganglion neurons: DOI: 10.1177/1744806916646111 mpx.sagepub.com A transcriptomic comparison Kathleen Yin, BPharm1,2, Gregory J Baillie, PhD1 and Irina Vetter, PhD1,2 Abstract Background: Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results: In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH- SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmye- linated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions: This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron- derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. Keywords Dorsal root ganglions, SH-SY5Y, F-11, ND7/23, transcriptomic analysis, molecular expression Date received: 11 January 2016; revised: 24 February 2016; accepted: 16 March 2016 Background properties defining subsets of peripheral sensory neurons, Peripheral sensory neurons enable humans to sense the as well as the G-protein coupled receptors (GPCRs) and world around us. Sensory nerve endings innervating the ion channels contributing to sensory neuron physiology skin, muscle, and viscera are responsible for detecting and pathophysiology. For example, the transient recep- external stimuli such as touch, vibration, temperature, tor potential (TRP) channels including TRPV1, TRPA1, and pain, which are then relayed to the spinal cord and and TRPM8 are activated by temperature and reactive the central nervous system. Peripheral sensory neurons chemicals, while GPCRs such as protease-activated are a heterogeneous group of neurons that can be clas- sified according to axonal transmission speed, degrees of 1 1 Centre for Pain Research, Institute for Molecular Bioscience, University of myelination, and functional characteristics. The cell Queensland, Queensland, Australia bodies of these pseudo-unipolar neurons are located in 2Pharmacy Australia Centre of Excellence, University of Queensland, the dorsal root ganglia (DRG) and are used extensively Queensland, Australia to study mammalian sensory processes, peripheral mech- anisms of pain, and the effect of drugs on the peripheral Corresponding author: Irina Vetter, Centre for Pain Research, Institute for Molecular Bioscience, nervous system. In recent years, significant advances 306 Carmody Road, Building 80, University of Queensland, St Lucia, have been made in unravelling the molecular and Queensland 4072, Australia. pharmacological basis of the diverse functional Email: [email protected] Creative Commons Non Commercial CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https:// us.sagepub.com/en-us/nam/open-access-at-sage). 2 Molecular Pain 0(0) receptors, histamine receptors, and bradykinin receptors interpretation of results obtained using SH-SY5Y, are activated by inflammatory mediators. While primary F-11, and ND7/23 cells. DRG neurons are used extensively to study the pharma- cological mechanisms involved in sensory neuron func- tions such as pain and itch, this approach has several Methods drawbacks. The isolation and culture of DRG neurons Animals is time consuming, highly technical, and requires the use of animals. Importantly, not all neurons survive this pro- Male wild-type C57BL/6 mice age six to eight weeks and cess.2 Removal of DRG neurons from the in vivo envir- weighing 20–23 g were used in the study. Ethical onment and axotomy of the peripheral processes also approval for experiments involving animal tissues was leads to adaptive changes such as down-regulation of obtained from the University of Queensland animal the potassium channel subunit KV9.1, overexpression ethics committee. Experiments were conducted in of the nerve growth factor receptor TrkA, and increased accordance with the Animal Care and Protection neuronal excitability.3–6 In addition, as DRG neurons Regulation Qld (2012), the Australian Code of Practice are heterogeneous, only a subset of cells is likely to for the Care and Use of Animals for Scientific Purposes, express the target of interest and a substantial proportion 8th edition (2013), and the International Association of cultured cells include non-neuronal cell types such as for the Study of Pain Guidelines for the Use of fibroblasts and glial cells. Accordingly, researchers have Animals in Research. All animals were obtained from turned to DRG-derived immortal cell lines as substitutes University of Queensland Biological Resources for intact DRG neurons when investigating endogenous (Brisbane, Australia). receptors. Common candidates are the DRG-hybridoma cell lines F-11 and ND7/23, which were derived from Chemicals embryonic or neonatal rat DRG neurons by fusion with the mouse neuroblastoma cell line N18Tg27,8 and The following compounds were obtained from Life display neuron-like properties including excitable mem- Technologies (Mulgrave, Victoria, Australia): L-gluta- branes and sensory neuronal cell surface markers. mine, Dulbecco’s Modified Eagle Medium (DMEM), Similarly, we have recently shown that the neuroblast- Ham’s F12 media, Roswell Park Memorial Institute oma cell line SH-SY5Y, which is commonly used as a medium, 0.25% trypsin/EDTA, foetal bovine serum model of adrenergic or dopaminergic neurons, exhibits (FBS), and phosphate buffered saline (PBS). The follow- traits of peripheral sensory neurons such as functional ing compounds were obtained from Sigma-Aldrich expression of the sensory neuron-specific sodium channel (Castle Hill, New South Wales, Australia): HAT media 9 TM isoform NaV1.7. However, although these cell lines are supplement Hybri-Max , capsaicin, collagenase, prote- often considered suitable models to study mechanisms of ase, menthol, and allyl isothiocyanate (AITC). Oxytocin nociceptor activation and individual investigations of and vasopressin were a kind gift from Professor Paul specific protein families have been conducted,9–11 a sys- Alewood (Institute for Molecular Bioscience, The tematic analysis of the expression profile of these cell University of Queensland). Calcium 4 No Wash dye lines in comparison with DRG neurons is lacking. was purchased from Molecular Devices (Sunnyvale, In this study, we conducted RNA-Seq analysis with CA, USA). particular emphasis on ion channel and GPCR expres- sion in DRG neuron-derived cell lines including the Cell culture human neuroblastoma cells SH-SY5Y,12,13 the mouse neuroblastoma/rat embryonic DRG neuron hybrid cell Human neuroblastoma SH-SY5Y cells (The European line F-11,8 and the mouse neuroblastoma/rat neonatal Collection of Cell Cultures) were grown in Roswell DRG neuron hybrid cell line ND7/23.7 Data from cell Park Memorial Institute medium with 15% FBS and lines were then compared to RNA-Seq analysis con- 2 mM L-glutamine and kept at 37 C/5% CO2; 0.25% ducted on whole murine DRG. Functional expression trypsin/EDTA was used to passage the cells every three of selected targets was confirmed by Ca2þ imaging. to five days at a 1:5 dilution ratio or when approximately While a number of sensory neuron-specific targets 90% confluent. including NaV1.7 were expressed in all cell lines, we Mouse neuroblastoma/rat embryonic DRG neuron also found several discrepancies, and none of the cell hybrid F-11 cells (The European Collection of Cell lines displayed an expression profile consistent with spe- Cultures) were grown in Ham’s F12 media with 10% cific subclasses of sensory neurons. This stresses the need FBS, 100 mM hypoxanthine, 0.4 mM aminopterin, and for further exploration on the functional receptors pre- 16 mM thymidine (HAT media supplement Hybri- TM sent on neuronal cell lines used as model DRG neurons, Max ) and kept at 37 C/5% CO2; 0.25% trypsin/ and highlights the importance for cautious EDTA was used to passage the cells every two to three Yin et al. 3 days at a 1:5 dilution ratio or when approximately 80% with Tophat with default analysis parameters in single- confluent. end mode. The number of mismatches were restricted to Mouse neuroblastoma/rat DRG neuron hybrid ND7/ no more than two bases. Reads from SH-SY5Y cells were 23 cells (The European Collection of Cell Cultures) were mapped against the human genome (hg19), DRG reads grown in DMEM with 10% FBS, 2 mM L-glutamine, were mapped against the mouse genome (mm9), and F-11 pyridoxine, and 110 mg/ml sodium pyruvate and kept and ND7/23 reads were mapped against both the mouse at 37 C/5% CO2; 0.25% trypsin/EDTA was used to pas- genome (mm9) and rat genome (rn5). Transcripts assem- sage the cells every three to five days at a 1:5 dilution bly was performed by Cufflinks with the reference anno- ratio or when approximately 90% confluent. tation-based assembly option14 using the UCSC annotation, and multi-reads correction was used.
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