Name: differentiation of the sh-sy5y human neuroblastoma cell line
Uploaded: 2016-02-17T13:00:00.000+00:00
Duration: 8 min 7 s
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 throughout cultures of SH-SY5Y cells.</li>
	<li>Prior to use, allow media to warm and equilibrate in an incubator to establish a proper pH balance before every step. For example, 50 ml of media takes approximately one hour to fully equilibrate (pH 7, 37 &#176;C, 5% CO2). 
	Note: This protocol uses a two-step splitting procedure that requires partially differentiated SH-SY5Y cells to be trypsinized and re-plated. This is a stressful process for these exceptionally fragile cells. Therefore, it is important to incubate the cells in trypsin for a minimal amount of time. This will allow for the preferential lift-off of neurons, leaving epithelial-like cells still attached to the dish.</li>
	<li>Perform trituration of differentiated cells slowly with a 10 ml plastic pipet with the tip against the bottom of the conical tube containing the cells. Perform trituration at a slow speed, up and down no more than five times.</li>
</ol>
2. Passage of SH-SY5Y Maintenance Cultures
<ol>
	<li>Split maintenance cultures when cells have reached 70-80% confluency, and do not exceed 10 to 15 passages. Cultures typically need to be passaged every 3-5 days (assuming cultures are not diluted more than 5-fold during splitting).</li>
	<li>To passage cells from a T-75 flask, aspirate off media, then rinse with approximately 10 ml 1x PBS. 
	Note: We do not recommend splitting the SH-SY5Y maintenance cultures further than 1:5 during passaging because this can cause the cells to die due to low confluency.</li>
	<li>Aspirate PBS, and then add 2.5 ml 0.05% Trypsin-EDTA (1x).</li>
	<li>Incubate in incubator 2-3 min and tip gently to release cells from the surface of the flask.</li>
	<li>Add 10 ml Basic Growth Media (see Table 2) and triturate 1-2 times.</li>
	<li>Spin down for 2 min at 1,000 x g, aspirate media, then resuspend pellet in 5 ml Basic Growth Media.</li>
	<li>Dilute cells from 1:3 to 1:5 in a total volume of 20 ml for normal plating in T-75 flask, or count and plate for differentiation (section 5).</li>
</ol>
3. Freezing SH-SY5Y Cells
<ol>
	<li>Freeze early passages of SH-SY5Y neuroblastoma cells in Basic Growth Media supplemented with 5% (v/v) DMSO.</li>
	<li>Initially, freeze aliquots at -80 &#176;C for 24 hr, then transfer to liquid nitrogen for long-term storage. 
	Note: For reference, a confluent (75-85%) T-75 flask will yield five 1 ml aliquots of SH-SY5Y cells for freezing. Each of these aliquots should contain anywhere from 2-5 million cells total.</li>
</ol>
4. Thaw and Culture Undifferentiated SH-SY5Y Neuroblastoma Cells
<ol>
	<li>Prepare Basic Growth Media.</li>
	<li>Rapidly thaw frozen cells in a 37 &#176;C water bath (approximately 2 min).</li>
	<li>Resuspend cells in 9 ml Basic Growth Media in a 15 ml conical tube, and then centrifuge for 2 min at 1,000 x g.</li>
	<li>Aspirate the supernatant while being careful not to disturb the pelleted cells, and gently resuspend cells in 10 ml Basic Growth Media.</li>
	<li>Plate cells onto a T-25 flask or 60 mm2 dish.</li>
	<li>The next day, replace media to remove dead cells.</li>
</ol>
5. Day 0: Plating Cells for Differentiation
<ol>
	<li>See Figure 1 for the differentiation schedule.</li>
	<li>Rinse undifferentiated cells with 1x PBS, aspirate, and then trypsinize using 1-2 ml warmed 1x 0.05% Trypsin-EDTA.</li>
	<li>When cells are in trypsin, incubate for approximately 3 min in an incubator.</li>
	<li>Quench the trypsin by adding 10 ml Basic Growth Media, rinse the sides of the flask or dish, and gently triturate 1-3 times. Transfer contents to a 15 ml conical tube.</li>
	<li>Centrifuge for 2 min at 1,000 x g, and aspirate the media while being careful not to disturb the pellet.</li>
	<li>Resuspend pellet in 5 ml Basic Growth Media and triturate 1-3 times.</li>
	<li>Count cells using a hemocytometer, then dilute using Basic Growth Media to 50,000 cells/ml.</li>
	<li>Plate 2 ml of cells per 35 mm2&#160;dish for a total of 100,000 cells per dish and place back into incubator.</li>
</ol>
6. Day 1: Change Media (Differentiation Media #1)
<ol>
	<li>Aliquot 50 ml of Differentiation Media #1 (see Table 2)and incubate in a 37 &#176;C water bath.</li>
	<li>When media is warmed, allow it to equilibrate in an incubator (37 &#176;C, 5% CO2) for at least one hr to establish a proper pH balance prior to use.</li>
	<li>Add Retinoic Acid (RA) (see Table 1) to warmed and equilibrated media immediately before adding media to dishes. 
	Note: Retinoic acid is light sensitive and should be stored in dark bottles at 4 &#176;C</li>
	<li>Gently aspirate off old media and discard.</li>
	<li>Add 2 ml Differentiation Media #1 with RA per 35 mm2&#160;dish and return to incubator.</li>
</ol>
7. Day 3: Change Media (Differentiation Media #1)
<ol>
	<li>Repeat Section 6 (steps 1-5)</li>
</ol>
8. Day 5: Change Media (Differentiation Media #1)
<ol>
	<li>Repeat Section 6 (steps 1-5)</li>
</ol>
9. Day 7: Split Cells 1:1
<ol>
	<li>Add RA to warmed and equilibrated Differentiation Media #1 immediately before adding media to dishes.</li>
	<li>Gently aspirate off old media and discard.</li>
	<li>Add 200 &#181;l warmed 0.05% 1x Trypsin EDTA per 35 mm2&#160;dish and warm in incubator approximately 2-3 min or until cells are visibly lifted from the plate as observed under a microscope.</li>
	<li>Quench the trypsin by adding 2 ml Differentiation Media #1 with RA per 35 mm2&#160;dish and use the media to rinse remaining neuronal cells off the plate. Then transfer contents to a 50 ml conical tube. 
	Note: During trypsinization steps, do not trypsinize too many dishes at once. This helps to ensure neuronal cultures are not incubated in trypsin for too long, which can be cytotoxic.</li>
	<li>Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipet.</li>
	<li>Aliquot 2 ml cell suspension into fresh 35 mm2&#160;dishes and return to incubator.</li>
</ol>
10. Day 8: Change Media (Differentiation Media #2)
<ol>
	<li>Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.</li>
	<li>Gently aspirate off old media and discard.</li>
	<li>Slowly add 2 mL Differentiation Media #2 (see Table 2) with RA per 35 mm2&#160;dish and return to incubator. Do not allow neurons to be exposed to air for an extended period of time as they can dry out quickly.</li>
</ol>
11. Day 9: Prepare Extracellular Matrix (ECM) Coated Dishes
<ol>
	<li>Thaw one vial of ECM solution on ice and dilute 1:100 into cold DMEM.</li>
	<li>Dispense 2 ml of mixture into each 35 mm2&#160;dish and ensure the entire base of the dish is covered.</li>
	<li>Place in an incubator (37 &#176;C, 5% CO2) for 1 hr or overnight.</li>
	<li>Aspirate mixture and allow to air dry for approximately 1 hr in a hood. Store at room temperature for up to 2 months.</li>
</ol>
12. Day 10: Transfer Cells onto ECM Coated Plates 1:1
<ol>
	<li>Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.</li>
	<li>Gently aspirate off media and discard.</li>
	<li>Add 200 &#181;l warmed trypsin to each 35 mm2&#160;dish and allow to incubate at room temperature for approximately 1-2 min or until neurons are visibly lifted from the dish as observed under a microscope. 
	Note: Execute this trypsinization step at room temperature so as not to over-incubate neurons with trypsin and cause damage. Neurons release from plates much faster than epithelial-like cells at this stage.</li>
	<li>Quench the trypsin by adding 2 ml Differentiation Media #2 per 35 mm2&#160;dish and use the media to rinse remaining neuronal cells off the plate. Then transfer contents to a 50 ml conical tube.</li>
	<li>Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipet.</li>
	<li>Dispense 2 ml cell suspension into ECM-coated 35 mm2&#160;dishes and return to incubator.</li>
</ol>
13. Day 11: Change Media (Differentiation Media #3)
<ol>
	<li>Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.</li>
	<li>Gently aspirate off old media and discard.</li>
	<li>Slowly add 2 ml Differentiation Media #3 (see Table 2) with RA per 35 mm2&#160;dish and return to incubator. Do not allow neurons to be exposed to air for an extended period of time.</li>
</ol>
14. Day 14: Change Media (Differentiation Media #3)
<ol>
	<li>Repeat Section 13 (steps 1-3)</li>
</ol>
15. Day 17: Last Media Change (Differentiation Media #3)
<ol>
	<li>Repeat Section 13 (steps 1-3)</li>
</ol>
16. Day 18: Neuronal Cultures Ready to Use
<ol>
	<li>Change media to fresh Differentiation Media #3 with RA every 3 days to maintain neuron health. 
	Note: Cells should be differentiated into neurons and exhibit a neuronal phenotype. Cultures are typically stable for up to 14 days following terminal differentiation, however duration of neuron viability is dependent on passage number of the undifferentiated cells at the start of differentiation. Higher passage numbers yield differentiated neurons with a shorter useful lifetime.</li>
</ol>

The authors have nothing to disclose.

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. This cell line begins as a heterogeneous population of adherent and suspended cells. This protocol aims to preserve both populations by withholding PBS washes before passaging or media change, but some loss of suspended cells is necessary to allow removal of the dead epithelial cells. The presented method produces a homogeneous population of differentiated human SH-SY5Y neurons for further experimentation.
Although other agents can be used to guide the differentiation of neurons into a cholinergic or adrenergic phenotype16,17, the use of RA to differentiate SH-SY5Y cells has been used previously to produce neurons with a dopaminergic phenotype37,38. Addition of RA has been shown to induce cellular differentiation through a number of mechanisms, including arresting cell cycle progression out of G0/G1, increasing expression of the cyclin-dependent kinase (CDK) inhibitors p21 and p27Kip1 and the anti-apoptotic proteins Bcl-2 and Bcl-xL, and enhancing PI3K/AKT activity which plays a role in neurite development and differentiation39.
It is imperative throughout the execution of this protocol to use gentle and sterile handling techniques, as SH-SY5Y cells are very sensitive to sudden change. It is because of this sensitivity that the gradual serum deprivation protocol is preferred over protocols that require making rapid changes in media composition. When splitting cells on days 7 and 10, it is important to minimize the amount of time that the partially differentiated neurons spend in trypsin. This reduces the probability of damaging neurons and promotes the preferential release of neurons versus epithelial cells, which take longer to detach. This helps to establish a more homogeneous neuronal population of cells and to minimize contamination with the proliferating and undifferentiated epithelial cells. It is also important to note reagents used for the culture and differentiation of SH-SY5Y cells should be replaced routinely and not be kept indefinitely. For example, Basic Growth Media can be kept up to 6 weeks while Differentiation Media should only be kept up to 2 weeks. This ensures reagents such as dbcAMP and BDNF are fresh and stable. Additionally, prepared RA should only be stored for up to 6 weeks and in the dark before a fresh batch should be made. We have observed greater consistency of neuronal outcomes with these precautions.
Once cells have been differentiated into mature neurons, they can be maintained for up to 2 weeks post-terminal differentiation and used for experimentation. Following terminal differentiation, media should be changed every 3-4 days (Differentiation Media #3). In contrast to differentiated neurons, maintenance flasks containing undifferentiated SH-SY5Y cell cultures may be kept over many weeks with regular passaging. It is important to passage maintenance cultures regularly, to prevent over-population of epithelial-like cells that are no longer capable of differentiating into neurons. When these cells outnumber those with neuro-potential, serum starvation will cause disproportionate amounts of cell death. Undifferentiated cultures can only be maintained for up to approximately 15 passages. Once the culture has exceeded around 15 passages, undifferentiated cells begin to die and have a rough, unhealthy appearance. Additionally, differentiation of high passage SH-SY5Y cells is more difficult as fewer cells will survive each split, and those that do often aggregate both cell bodies and axons, making subsequent analyses more difficult.
There is a distinct decrease in cell number during the differentiation process as many cells are lost or do not survive the splitting process. Therefore, at the beginning of any experiment involving differentiated SH-SY5Y cells, one should account for ~30-40% loss of neurons and ensure a proper number of cells is plated at the start to achieve the desired yield. While plating 100,000 cells produces plenty of healthy, mature SH-SY5Y neurons, fewer or larger numbers may be initially plated depending on experimental need.
It is clear that fully differentiated SH-SY5Y neurons provide a closer approximation of mature human neurons found in vivo than do their undifferentiated progenitor cell counterparts (Figure 2). These neurons will provide an advantageous model for future characterizations of their neurobiology, for the study of neurotropic viruses, or to screen for chemotherapeutic toxicity in neurons.

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><tbody><tr><td>B-27</td><td>ThermoFisher Scientific</td><td>17504-044</td><td>See &lt;strong&gt;Table 1&lt;/strong&gt; for preparation</td></tr><tr><td>Brain-Derived Neurotrophic Factor (BDNF)</td><td>Sigma</td><td>SRP3014 (10 &amp;mu;g)/B3795 (5 &amp;mu;g)</td><td>See &lt;strong&gt;Table 1&lt;/strong&gt; for preparation</td></tr><tr><td>dibutyryl cyclic AMP (db-cAMP)</td><td>Sigma</td><td>D0627</td><td>See &lt;strong&gt;Table 1&lt;/strong&gt; for preparation</td></tr><tr><td>DMSO</td><td>ATCC</td><td>4-X</td><td>-</td></tr><tr><td>Minimum Essential Medium Eagle (EMEM)</td><td>Sigma</td><td>M5650</td><td>-</td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Hyclone</td><td>SH30071.03</td><td>See &lt;strong&gt;Table 1&lt;/strong&gt; for preparation</td></tr><tr><td>GlutamaxI</td><td>Life Technologies</td><td>35050-061</td><td>-</td></tr><tr><td>Glutamine</td><td>Hyclone</td><td>SH30034.01</td><td>-</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>BP366-1</td><td>See &lt;strong&gt;Table 1&lt;/strong&gt; for preparation</td></tr><tr><td>MaxGel Extracellular Matrix (ECM) solution</td><td>Sigma</td><td>E0282</td><td>See step 11 of the protocol</td></tr><tr><td>Neurobasal</td><td>Life Technologies</td><td>21103-049</td><td>-</td></tr><tr><td>Penicillin/Streptomycin (Pen/Strep)</td><td>Life Technologies</td><td>15140-122</td><td>-</td></tr><tr><td>Retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td>Should be stored in the dark at 4 &amp;deg;C because this reagent is light sensitive</td></tr><tr><td>SH-SY5Y Cells</td><td>ATCC</td><td>CRL-2266</td><td>-</td></tr><tr><td>0.5% Trypsin + EDTA</td><td>Life Technologies</td><td>15400-054</td><td>-</td></tr><tr><td>Falcon 35 mm TC dishes</td><td>Falcon (A Corning Brand)</td><td>353001</td><td>-</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 best possible representation of what may be occurring in neurons in vivo. The above protocol yields highly viable, homogenous, differentiated neuronal cultures within 18 days, that can be used for subsequent biochemical and imaging analyses. Undifferentiated SH-SY5Y neuroblastoma cells demonstrate a large, flat, epithelial-like phenotype with numerous short processes extending outward (Figure 2A), while differentiated cells possess several neuritic projections that connect to surrounding cells (Figure 2B). It is important that when differentiating SH-SY5Y cells, cells are incubated with trypsin for a minimal length of time to ensure that only neurons are released from the dish. This leaves behind the undifferentiated epithelial cells that would otherwise contaminate the differentiated neuronal cell population. The neuronal characteristics of fully differentiated SH-SY5Y cells are demonstrated by techniques such as immunofluorescence detection of classical neuronal markers (Figure 3).
SH-SY5Y cells demonstrate a variety of different phenotypes during the course of differentiation and it is important to be able to identify healthy neurons from those that are stressed. On differentiation day 1, prior to the introduction of Differentiation Media #1, cells have a flat, retracted phenotype with short, stubby processes (Figure 4A). Following 5 days of serum deprivation, SH-SY5Y cells begin to develop longer projections and demonstrate a more neuronal phenotype (Figure 4B). Passaging is a harsh process for SH-SY5Y cells, and on days immediately following passaging, cells appear unhealthy. This is evidenced by cell body clumping and the presence of fewer, shorter processes. (See before images: Figures 4C and 4E, and after images: Figures 4D and 4F). Approximately 48 hr following a split, cells appear to recover, and fully mature, differentiated neurons are obtained at Day 18 (Figure 2B). This is evidenced by a reduction in cell body clumping, and extension of numerous thin, branched neuritic processes that often connect to neighboring cells.
Factors that are essential to obtaining reproducible and viable neuronal cultures include using heat-inactivated FBS, minimizing trypsin incubation time, and gentle trituration. Importantly, this protocol details the use of four different media formulations with various concentrations of hiFBS, thereby creating a gentle transition into a serum-starved state for the cells. When mature SH-SY5Y cells are healthy, they demonstrate numerous projections that connect to surrounding neurons (Figure 5A). It should be noted that a distinctive epithelial phenotype will overtake the neuronal cultures if they are mishandled during the differentiation process (Figure 5B). Additionally, if neurons begin to die, neurites will retract, cell bodies will begin to clump together and round up, and debris from neurite degeneration will start to accumulate at and around cell processes. This process is akin to what is demonstrated in Figures 5C and 5D. While Figure 5C shows a more natural progression of cell death following nutrient and environmental deprivation, Figure 5D shows differentiated SH-SY5Y neurons 4 hr following infection with an HSV-1 strain, KOS, at a multiplicity of infection (MOI) of 106 PFU. This protocol has been thoroughly optimized in the lab and yields reproducible, homogeneous populations of neurons, which are essential for downstream analyses and experimentation.
<img alt="Figure 1" src="/files/ftp_upload/53193/53193fig1.jpg" /> 
Figure 1: Timetable of differentiation procedure. The differentiation process consists of 11 steps spread out over the course of an 18 day period. On the first day of the differentiation protocol (day 0), between 25,000 and 100,000 cells are plated onto uncoated 35 mm dishes. On days 1, 3, and 5, old media is removed and Differentiation Media #1 is applied. On day 7, cells are split 1:1 onto uncoated 35mm dishes in Differentiation Media #1. On day 8, the media is changed to Differentiation Media #2, and on day 10, cells are again split 1:1, but this time onto ECM-coated 35 mm dishes in Differentiation Media #2. On days 11, 14, and 17, old media is removed and Differentiation Media #3 is applied. On day 18, differentiated neurons are ready to use for downstream applications.
<img alt="Figure 2" src="/files/ftp_upload/53193/53193fig2.jpg" /> 
Figure 2: Morphological appearance of undifferentiated and differentiated SH-SY5Y cells. (A) Undifferentiated SH-SY5Y cells have a flat phenotype with few projections while (B) differentiated SH-SY5Y neurons demonstrate extensive and elongated neuritic projections. Images were obtained in phase at 20X magnification using an inverted epifluorescence microscope.
<img alt="Figure 3" src="/files/ftp_upload/53193/53193fig3.jpg" /> 
Figure 3: Markers of neuronal differentiation.&#160;Immunofluorescence illuminates neuronal features of fully differentiated SH-SY5Y cells. (A) Anti-SMI31 (green) stains the phosphorylated neurofilament H in the extensive network of neurites. (B) Anti-MAP2 (red) labels microtubule-associated protein 2, revealing the neuronal soma and proximal portion of neurites. Corresponding phase image for each immunofluorescence panel is shown to the right. Images were obtained at 10X magnification using an inverted epifluorescence microscope. Scale bar, 100 &#956;m.
<img alt="Figure 4" src="/files/ftp_upload/53193/53193fig4.jpg" /> 
Figure 4: Intermediate steps of the differentiation protocol. (A) Day 1 of differentiation. Cells maintain a retracted phenotype with short projections. (B) Day 5 of differentiation. Cells have been exposed to 5 days of serum deprivation (Differentiation Media #1). The surviving cells begin to elongate and form longer processes that connect with neighboring cells. (C) Day 7 of differentiation prior to split. Cells demonstrate high numbers of long processes, with fewer numbers of cells demonstrating an epithelial-like phenotype. (D) Day 8 of differentiation, one day after the first passage. Following splitting, cell bodies form clumps and processes appear short as a result of the passaging procedure. (E) Day 10 of differentiation, prior to split onto ECM-coated plates. Cells demonstrate longer processes that make connections with nearby cells. Cell body clustering is also evident. (F) Day 11 of differentiation, one day following second split. Cells are stressed following the second passage and many neurons are ultimately lost. However, the remaining population is viable, homogenous, and neuronal in phenotype. Cell bodies produce larger clusters and processes begin to emit from the base of the clusters.
<img alt="Figure 5" src="/files/ftp_upload/53193/53193fig5.jpg" /> 
Figure 5: Epithelial overgrowth and neuronal death - two alternative outcomes of the differentiation process. (A) Healthy, mature SH-SY5Y neurons demonstrate diffuse axonal projections connecting to neighboring cells. (B) In some instances, cells with a more epithelial-like phenotype overtake maintenance cultures. This over-population of epithelial-like cells may be due to infrequent passaging of maintenance cultures. These cultures should be discarded, as the epithelial-like cells will continue to outnumber neurons. Arrows denote epithelial-like cells. (C) When SH-SY5Y cells are unhealthy and begin to die, cell bodies round up and processes degrade, generating a significant amount of debris. (D) Noticeable accumulation of cellular debris and retraction of neuronal processes is evident in mature, differentiated neurons, 4 hr following infection with a strain of herpes simplex virus 1 (HSV-1) KOS at a multiplicity of infection (MOI) of 106 PFU.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Details</td>
			<td>Stock</td>
			<td colspan="5">Instructions</td>
		</tr>
		<tr>
			<td rowspan="2">10 &#956;M RA</td>
			<td>All-trans retinoic acid</td>
			<td rowspan="2">5 mM</td>
			<td colspan="5" rowspan="2">Resuspend 50 mg RA in 33.3 ml 95% EtOH.&#160; RA is sensitive to heat, light, and air.&#160; Keep in a dark bottle and store at 4 &#176;C for up to 6 weeks.&#160; Use at 1:500 dilution and dilute into differentiation media immediately prior to use</td>
		</tr>
		<tr>
			<td>(300.44 g/mol)</td>
		</tr>
		<tr>
			<td>1x B-27</td>
			<td>B-27 Supplement</td>
			<td>50x</td>
			<td colspan="5">Thaw 1-10 ml bottle and aliquot remainder into single-use 1 ml aliquots and store at -80 &#176;C.&#160; Store 10 ml bottles at -20 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">20 mM KCl</td>
			<td>Potassium Chloride</td>
			<td rowspan="2">1 M</td>
			<td colspan="5" rowspan="2">Add 250 ml water to 18.6 g KCl and sterile filter.&#160; Store at room temperature</td>
		</tr>
		<tr>
			<td>(74.55 g/mol)</td>
		</tr>
		<tr>
			<td rowspan="2">2 mM db-cAMP</td>
			<td>dibutyryl cyclic AMP</td>
			<td rowspan="2">1 M</td>
			<td colspan="5" rowspan="2">Resuspend full bottle by adding 2.04 ml water to 1 g db-cAMP.&#160; Sensitive to light and moisture.&#160; Store in aliquots of 100 &#956;l or 200 &#956;l at either -20 &#176;C or -80 &#176;C</td>
		</tr>
		<tr>
			<td>(491.37 g/mol)</td>
		</tr>
		<tr>
			<td>50 ng/ml BDNF</td>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>-</td>
			<td colspan="5">Centrifuge vial to get powder to bottom.&#160; Resuspend 10 &#956;g vial in 1 ml Neurobasal + 1x B27 or 5 &#956;g vial in 0.5 ml Neurobasal + 1x B27 to get 10 &#956;g/ml.&#160; Use at 1:200 dilution.&#160; Store working aliquots at -80 &#176;C (ex: 250 &#956;l)</td>
		</tr>
		<tr>
			<td>hiFBS&#160;</td>
			<td>Heat-inactivated Fetal Bovine Serum</td>
			<td>-</td>
			<td colspan="5">Aliquot thawed FBS into 50 ml conical tubes.&#160; Heat at 56 &#176;C in a water bath for 30 min.&#160; Remove and freeze working aliquots at -20 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Stock solutions and components.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="3">Basic Growth Media</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume for 500 ml</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>EMEM</td>
			<td>415 ml EMEM</td>
			<td></td>
		</tr>
		<tr>
			<td>15% hiFBS</td>
			<td>75 ml hiFBS</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Pen/Strep</td>
			<td>5 ml Pen/Strep</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>2 mM Glutamine</td>
			<td>5 ml Glutamine</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td colspan="2">*Keep for 6 weeks maximum</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Differentiation Media #1</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume for 50 ml</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>EMEM</td>
			<td>48 ml EMEM</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% hiFBS</td>
			<td>1.3 ml hiFBS</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Pen/Strep</td>
			<td>500 &#956;l Pen/Strep</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>2 mM Glutamine</td>
			<td>500 &#956;l Glutamine</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>10 &#956;M RA</td>
			<td>100 &#956;l RA (5mM stock)</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="3">*Keep for 2 weeks maximum and add RA immediately prior to use</td>
		</tr>
		<tr>
			<td colspan="3">*Do not keep extra media once RA is added - RA is unstable</td>
		</tr>
		<tr>
			<td colspan="3">Differentiation Media #2</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume for 50&#160;ml</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>EMEM</td>
			<td>49 &#160;ml&#160;&#160;EMEM</td>
			<td></td>
		</tr>
		<tr>
			<td>1% hiFBS</td>
			<td>500 &#956;l&#160;&#160;hiFBS</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Pen/Strep</td>
			<td>500 &#956;l&#160;Pen/Strep</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>2 mM Glutamine</td>
			<td>500 &#956;l&#160;&#160;Glutamine</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>10 &#956;M RA</td>
			<td>100 &#956;l&#160;&#160;RA (5mM stock)</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="3">*Keep for 2 weeks maximum and add RA immediately prior to use</td>
		</tr>
		<tr>
			<td colspan="3">*Do not keep extra media once RA is added - RA is unstable</td>
		</tr>
		<tr>
			<td colspan="3">Differentiation Media #3</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume for 50 ml</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>47 ml&#160;Neurobasal</td>
			<td></td>
		</tr>
		<tr>
			<td>1x B-27</td>
			<td>1 ml&#160;B-27 (50X stock)</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>20 mM KCl</td>
			<td>1 ml&#160;KCl (1M stock)</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>1x Pen/Strep</td>
			<td>500 &#956;l&#160;&#160;Pen/Strep</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>2 mM GlutamaxI</td>
			<td>500 &#956;l&#160;&#160;GlutamaxI (100x stock)</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>50 ng/ml BDNF</td>
			<td>250 &#956;l&#160;&#160;BDNF stock (10 &#956;g/ml)</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>2 mM dibutyryl cyclic AMP (db-cAMP)</td>
			<td>100 &#956;l&#160;&#160;db-cAMP (1M stock)</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>10 &#956;M RA</td>
			<td>100 &#956;l&#160;&#160;RA (5 mM stock)</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="3">*Keep for 2 weeks maximum and add RA immediately prior to use</td>
		</tr>
		<tr>
			<td colspan="3">*Do not keep extra media once RA is added - RA is unstable</td>
		</tr>
	</tbody>
</table>
Table 2: Media recipes.

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 ...

differentiation-of-the-sh-sy5y-human-neuroblastoma-cell-line

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

71.2K Views.  The Pennsylvania State University. 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. 

Video: Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

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 microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

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

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Neuroscience

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Cancer Research

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Genetics

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Bioengineering

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...