Name: differentiation of the sh-sy5y human neuroblastoma cell line
Uploaded: 2016-02-17T13:00:00
Description: Watch this Scientific Journal Video about Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line at JoVE.com

We are grateful for the contributions of Yolanda Tafuri in optimizing conditions for SH-SY5Y differentiation, and for the support of Dr. Lynn Enquist, in whose lab this work was initiated. Y. Tafuri contributed the images shown in Figure 3. This work was supported by the NIH-NIAID Virus Pathogens Resource (ViPR) Bioinformatics Resource Center (MLS and L. Enquist) and K22 AI095384 (MLS).

1. General Considerations
<ol>
	<li>See the Table of Materials/Equipment for a list of necessary reagents. Perform all steps under strict aseptic conditions.</li>
	<li>Use heat-inactivated fetal bovine serum (hiFBS) for all media preparations that include FBS. To heat-inactivate, warm a 50 ml aliquot of FBS at 56 &#176;C for 30 min, inverting every 10 min (see also Table 1). 
	Note: When FBS is used without heat-inactivation, the epithelial-like phenotype progresses more quickly t...

The above protocol provides a straightforward and reproducible method to generate homogenous and viable human neuronal cultures. This protocol utilizes techniques and practices that integrate several previously published methods1-4 and aims to delineate the best practices of each. Differentiation of SH-SY5Y cells relies on gradual serum deprivation; the addition of retinoic acid, neurotrophic factors and extracellular matrix proteins; and serial splitting to select for differentiated mature adherent neurons. T...

The ability to use in vitro model systems has greatly enhanced the fields of neurobiology and the neurosciences. Cells in culture provide an efficient platform to characterize protein functionality and molecular mechanisms underlying specific phenomena, to understand the pathology of disease and infection, and to perform preliminary drug testing assessments. In neurobiology, the major types of cell culture models include primary neuronal cultures derived from rats and mice, and neuroblastoma cell lines such as rat B35 cells5, Neuro-2A mouse cells6, and rat PC12 cells7. Although use of such cell lines has advanced the field significantly, there are several confounding factors associated with handling non-human cells and tissue. These include understanding species-specific differences in metabolic processes, phenotypes of disease manifestation, and pathogenesis when compared to humans. It is also important to note that there are significant differences between mouse and human gene expression and transcription factor signaling, highlighting the limitations of rodent models and the importance of understanding which pathways are conserved between rodents and humans8-11. Others have employed the use of human neuronal cell lines including the N-Tera-2 (NT2) human teratocarcinoma cell line and inducible pluripotent stem cells (iPSCs). These cell lines provide good models for in vitro human systems. However, differentiation of NT2 cells with retinoic acid (RA) results in the generation of a mixed population of neurons, astrocytes, and radial glial cells12, necessitating an additional purification step to obtain pure populations of neurons. Additionally, NT2 cells demonstrate a highly variable karyotype13, with greater than 60 chromosomes in 72% of cells. iPSCs demonstrate variability in differentiation between different cell lines and vary in differentiation efficiency14. It is therefore desirable to have a consistent and reproducible human neuronal cell model to complement these alternatives.
SH-SY5Y neuroblast-like cells are a subclone of the parental neuroblastoma cell line SK-N-SH. The parental cell line was generated in 1970 from a bone marrow biopsy that contains both neuroblast-like and epithelial-like cells15. SH-SY5Y cells have a stable karyotype consisting of 47 chromosomes, and can be differentiated from a neuroblast-like state into mature human neurons through a variety of different mechanisms including the use of RA, phorbol esters, and specific neurotrophins such as brain-derived neurotrophic factor (BDNF). Prior evidence suggests that the use of different methods can select for specific neuron subtypes such as adrenergic, cholinergic, and dopaminergic neurons16,17. This latter aspect makes SH-SY5Y cells useful for a multitude of neurobiology experiments.
Several studies have noted important differences between SH-SY5Y cells in their undifferentiated and differentiated states. When SH-SY5Y cells are undifferentiated, they rapidly proliferate and appear to be non-polarized, with very few, short processes. They often grow in clumps and express markers indicative of immature neurons18,19. When differentiated, these cells extend long, branched processes, decrease in proliferation, and in some cases polarize2,18. Fully differentiated SH-SY5Y cells have been previously demonstrated to express a variety of different markers of mature neurons including growth-associated protein (GAP-43), neuronal nuclei (NeuN), synaptophysin (SYN), synaptic vesicle protein II (SV2), neuron specific enolase (NSE) and microtubule associated protein (MAP)2,16,17,20, and to lack expression of glial markers such as glial fibrillary acidic protein (GFAP)4. In further support that differentiated SH-SY5Y cells represent a homogeneous neuronal population, removal of BDNF results in cellular apoptosis4. This suggests that survival of differentiated SH-SY5Y cells is dependent on trophic factors, similar to mature neurons.
Use of SH-SY5Y cells has increased since the subclone was established in 19783. Some examples of their use include investigating Parkinson&#39;s disease17, Alzheimer&#39;s disease21, and the pathogenesis of viral infection including poliovirus22, enterovirus 71 (EV71)23,24, varicella-zoster virus (VZV)1, human cytomegalovirus25, and herpes simplex virus (HSV)2,26. It is important to note that several studies using SH-SY5Y cells have used these cells in their undifferentiated form, especially in the field of neurovirology27-36. The difference in the observed phenotype of undifferentiated versus differentiated SH-SY5Y cells raises the question of whether the observed progression of infection would be different in mature differentiated neurons. For example, differentiated SH-SY5Y cells have a higher efficiency of HSV-1 uptake versus undifferentiated, proliferating SH-SY5Y cells, which may be due to a lack of surface receptors that bind HSV and modulate entry on undifferentiated SH-SY5Y cells2. It is therefore critical that when designing an experiment focused on testing neurons in vitro, SH-SY5Y cells should be differentiated in order to obtain the most accurate results for translation and comparison to in vivo models.
The development of a reliable method to generate human neuronal cultures is imperative to allow researchers to perform translational experiments that accurately model the human nervous system. The protocol presented here is a procedure that delineates best practices derived from previous methods1-4 to enrich for human neurons that are differentiated using retinoic acid.

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">B-27</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:133px;">17504-044</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:141px;">Brain-Derived Neurotrophic Factor (BDNF)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">SRP3014 (10ug)/B3795 (5ug)</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">dibutyryl cyclic AMP (db-cAMP)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">D0627</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">DMSO</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:133px;">4-X</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:141px;">Minimum Essential Medium Eagle (EMEM)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">M5650</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:141px;">Hyclone</td>
			<td style="width:133px;">SH30071.03</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">GlutamaxI</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">35050-061</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Glutamine</td>
			<td style="width:141px;">Hyclone</td>
			<td style="width:133px;">SH30034.01</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Potassium Chloride (KCl)</td>
			<td style="width:141px;">Fisher Scientific</td>
			<td style="width:133px;">BP366-1</td>
			<td style="width:232px;">See Table 1 for preparation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:141px;">MaxGel Extracellular Matrix (ECM) solution</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">E0282</td>
			<td style="width:232px;">See step 11 of the protocol</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Neurobasal</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">21103-049</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:141px;">Penicillin/Streptomycin (Pen/Strep)</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">15140-122</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">Retinoic acid (RA)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:133px;">R2625</td>
			<td style="width:232px;">Should be stored in the dark at 4&#176; C because this reagent is light sensitive</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">SH-SY5Y Cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:133px;">CRL-2266</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:141px;">0.5% Trypsin + EDTA</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:133px;">15400-054</td>
			<td style="width:232px;">-</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:141px;">Falcon 35mm TC dishes</td>
			<td style="width:141px;">Falcon (A Corning Brand)</td>
			<td style="width:133px;">353001</td>
			<td style="width:232px;">-</td>
		</tr>
	</tbody>
</table>

differentiation of the sh-sy5y human neuroblastoma cell line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

At present, there are many instances in the field of neurobiology and neurovirology where undifferentiated SH-SY5Y cells are being used as a functional model for human neurons27-36, and importantly, undifferentiated cells may lack phenotypes such as optimal viral uptake2 that are necessary for accurate interpretation. It is critical that when using SH-SY5Y cells or any other in vitro neuronal system, cells are appropriately differentiated into neurons, in order to obtain data that is the be...

Watch this Scientific Journal Video about Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line at JoVE.com

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Research

JoVE Journal

Developmental Biology

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...