Name: a neurite outgrowth assay and neurotoxicity assessment with human neural progenitor cell-derived neurons
Uploaded: 2020-08-06T15:40:00
Description: Watch this Scientific Journal Video about A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons at JoVE.com

This research was funded by NIMAD research grant (940714) awarded to MAF.

Ethics Statement: Fetal specimens were received from the Birth Defects Research Laboratory at the University of Washington in Seattle through a tissue distribution program supported by the National Institute of Health (NIH). The Birth Defects Research Laboratory obtained appropriate written informed consent from the parents and the procurement of tissues was monitored by the Institutional Review Board of the University of Washington. All the work was performed with approval by the Human Subject Research Office at the Uni...

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This protocol is one of the few published papers describing the test for neurite length upon treatment with test compounds. Furthermore, we describe how to use hNPCs for a neurite outgrowth assay and neurotoxicity assessment. By utilizing this neurite outgrowth assay and neurotoxicity assessment on hNPCs-derived neurons, the neurogenic potential of a category of epigenetic small-molecule compounds, HDAC inhibitors, in inducing neurite outgrowth is demonstrated22. Furthermore, in another paper pres...

Neurite growth is a process fundamental to the formation of the neuronal network and nerve regeneration1,2. Following an injury, neurite outgrowth plays a key role in regeneration of the nervous system. Neurite outgrowth is also an important element of the extracellular signaling in inducing neuronal regenerative activities to enhance the outcomes for neurodegenerative disorders and neuronal injury3,4,5,6.
By maintaining their differentiation potential in producing various neural lineages, human neural progenitor cells (hNPCs) could provide a model system for studies of central nervous system (CNS) function and development7,8,9. High translational potential and physiological relevance of hNPCs as a primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screenings. However, the maintenance and scaling of the primary cell models for high-throughput assays could be time-consuming and labor-intensive10,11,12,13.
In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment is another application using the hNPC-derived neurons. There are thousands of commercial chemical compounds that are either not examined or with poorly understood neurotoxicity potential. Therefore, more reliable and effective screening experiments to assess, distinguish, and rank compounds based on their potential to elicit developmental neurotoxicity is in high demand14. The increase in prevalence and incidence of neurological disorders along with the abundance of untested compounds in the environment necessitates the development of more trustworthy and efficient experiments to identify hazardous environmental compounds that may pose neurotoxicity15.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.

<table>
	<tbody>
		<tr>
			<td>4-well Glass Chamber Slides</td>
			<td>Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III</td>
			<td>Thermo</td>
			<td>MA1-118X</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 - minus vitamin A</td>
			<td>Thermo</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>PeproTech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A8531</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo</td>
			<td>Promega</td>
			<td>G7572</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell</td>
			<td>Corning</td>
			<td>432000</td>
			<td>Cell freezing containers ensuring standardized controlled-rate -1&#8451;/minute cell freezing in a -80&#8451; freezer</td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>StemCell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium containing 10% DMSO</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>Gibco</td>
			<td>PHG6015</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Thermo</td>
			<td>FB002</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanyl-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Thermo</td>
			<td>50062Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Calbiochem</td>
			<td>375095</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A92902-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>StemCell Technologies</td>
			<td>5854</td>
			<td>Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 384-Well Polystyrene White Microplates</td>
			<td>Thermo</td>
			<td>164610</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly&#8208;L&#8208;lysine</td>
			<td>Sigma</td>
			<td>P5899-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>Cell dissociation reagent containing proteolytic and collagenolytic enzymes</td>
		</tr>
		<tr>
			<td>Synaptophysin</td>
			<td>Thermo</td>
			<td>MA5-14532</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

a neurite outgrowth assay and neurotoxicity assessment with human neural progenitor cell-derived neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The protocol presented in the manuscript has successfully been used in two recently published papers22,23. Figure 3 demonstrates the use of hNPCs-derived neurons in examining the effect of HDAC inhibitors as epigenetic compounds on the extension of neurites as a marker for neurite outgrowth and subsequent neurogenic ability of small molecule compounds.
Furthermore, in Figure 4...

Watch this Scientific Journal Video about A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons at JoVE.com

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

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Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...