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. 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 ...

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Developmental Biology

70.8K 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 ...

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 membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

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 phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

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 methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

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 muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

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 a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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

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 to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

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 failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like 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 transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

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