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 ...

The overall goal of this protocol is to provide neurobiologists and neurovirologists with an easy, reproducible method to generate differentiated human neurons for the use in subsequent in vitro assays. This method can help to answer key questions in infectious disease research, such as determining the neuronal responses to infection or drug treatment. It can also be used to assess proteomic or genomic changes associated with specific experimental conditions.The main advantage of this technique is that it provides an easy to follow protocol that reproducibly yields homogenous cultures of fully differentiated human neurons. Generally, individuals new to this method will struggle, because of the sensitivity of these cells to distruptions, such as tripsinization, triteration, or exposure to suboptimal environmental conditions. To begin this procedure, allow the media to warm and equilibrate in the incubator, in order to establish a proper pH balance before use.Then, split the cultures of the maintenance phase when the cells reach 70 to 80%confluency. To passage the cells from a T-75 flask, aspirate off the media. Then, rinse the flask with 10 milliliters of 1X PBS.Next, aspirate the PBS, and add 2.5 milliliters of 0.05%or 1X tripsin EDTA. After that, incubate the cells for two to three minutes. Then, tip the flask gently to release the cells from the surface.Subsequently, add 10 milliliters of basic growth media and triterate the cells one to two times. Next, spin bound the cells for two minutes at 1000 times G.Afterward, aspirate the media. Resuspend the palate in five milliliters of basic growth media.For normal plating in the T-75 flask, dilute the cells to a total volume of 20 milliliters, or count and plate the cells for differentiation. Here is the schedule for differentiation. On day zero, count the cells using a hemocytometer.Then, dilute the cell suspension to 50, 000 cells per milliliter, using basic growth media. Plate two milliliters of cells per 35 millimeter dish, for a total of 100, 000 cells per dish. Subsequently, place the cells in the incubator at 37 degrees Celsius.On day one, add RA to the warmed and equilibrated media. Gently aspirate the old media and discard. Next, add two milliliters of differentiation media number one, with RA, to each 35 millimeter dish, and return to the incubator.On day seven, gently aspirate the old media and discard. Add 200 microliters of warmed tripsin EDTA to each 35 millimeter dish. Then, place the cells in the incubator for two to three minutes, or until they are visibly lifted from the plate, as observed under a microscope.Quench the tripsin by adding two milliliters of differentiation media number one, with RA to each 35 millimeter dish, and use the media to rinse the remaining neuronal cells off the plate. Then, transfer the full volume to a 50 millileter conical tube. Gently triterate up and down slowly with a 10 milliliter plastic pipette no more than five times.After that, aliquot two milliliters of the cell suspension into the fresh 35 millimeter dishes, before returning them to the incubator. On day 10, gently aspirate off the media and discard. Add 200 microliters of warmed tripsin to each 25 millimeter dish, and incubate at room temperature for approximately one to two minutes, or until the neurons are visibly lifted from the dish as observed under a microscope.Day 10 of the protocol is particularly difficult, because of the sensitivity of the cells at this stage of differentiation. It is imperative to minimize the time the cells are in tripsin to approximately one minute at room temperature, and then put the cells immediately back into the incubator. Then, quench the tripsin by adding two milliliters of differentiation media number two to each 35 millimeter dish, and use the media to rinse the remaining neuronal cells off the plate.Afterward, transfer the contents to a 50 milliliter conical tube. Gently triterate up and down slowly with a 10 milliliter plastic pipette, no more than five times. Dispense two milliliters of cell suspension into ECM-coated 35 millimeter dishes before returning them to the incubator at 37 degrees Celsius.On day 11, gently aspirate off the old media and discard. Slowly add two milliliters of differentiation media number three with RA to each 35 millimeter dish, before returning them to the incubator. On day 18, change the media to fresh differentiation media number three with RA once every three days to maintain neuron health.Do not allow neurons to be exposed to air for an extended period of time. After seven days of differentiation, there is an increase in the number of processes on the neuronal-like cells, and there are fewer epithelial-like cells present. Following the first passage, the neuronal cell bodies clump together, and their processes appear shorter, highlighting the stress that the cells encounter during tripsinization and replating.Prior to plating onto the ECM-coated plates on day 10, longer processes are evident on the differentiating neurons. On day 11, following the final passage, the neurons are visibly stressed. This can result in an apparent decrease in the total number of cells, and clumping of the remaining cell bodies.However, the cells will recover over the next seven days. On day 18, the cells are terminally differentiated into neurons that have healthy, elongated projections. While attempting this procedure, it's important to remember to limit the amount of time the cells are in tripsin or exposed to air.It is also important to ensure that all reagants are always prepared fresh. Following this procedure, other methods, such as infection with a pathogenic agent, can be performed to assess subsequent changes in morphology, transcript levels, or protein expression. Thanks for watching, and good luck.

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