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...

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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 ...

The presented protocol describing neurite outgrowth assay that is highly relevant to human biology and neurotoxicity assessment of a small molecule compounds. High translational potential and physiological relevance of human neural progenitor cells as the primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screening and neurotoxicity assessment. Utilizing the presented protocol, human induced pluripotent stem cell and human embryonic stem cell-derived neurons can also be used as to alternative cell sources for the neurite outgrowth assay and neurotoxicity assessment.Begin by collecting the media with floating neurospheres and transferring it to a 50 milliliter conical tube. Centrifuge the tube at 300 to 400 times g for three minutes. Then carefully aspirate the supernatant.and resuspend the spheres in 500 microliters of defrosted cell dissociation reagent. Incubate the spheres at 37 degrees Celsius for five to 15 minutes. Then add five to 10 milliliters of pre-warmed culture media and centrifuge the tube at 300 to 400 times g for five minutes to sediment the neurospheres.Aspirate the supernatant and add two milliliters of cultural media pipetting up and down with a 1000 microliter pipette until the neurospheres form a single cell suspension. After counting the cells, add two to 3 million cells to a T25 flask in 10 milliliters of culture media. Replace half the culture media every three days.Use commercially available cryopreservation medium to freeze the cells. Transfer the neurospheres to a 50 milliliter tube and allow the spheres to settle at the bottom. Use a 200 or 1000 microliter pipette tip to transfer the big neurospheres to a new 50 milliliter tube before passaging.Centrifuge the remaining neurospheres at 300 to 400 times g for three minutes and carefully remove the supernatant. Resuspend up to a thousand spheres in one milliliter of cryopreservation reagent and transfer it to a cryo tube. Store the tube overnight at minus 80 degrees Celsius, then move it to liquid nitrogen for long-term storage.To measure neuronal outgrowth, open the image in Fiji and select Analyze, Tools and ROI Manager. Right-click on the fifth icon in the tool bar Straight and switch to Freehand Line. Optionally, double-click on the same icon to change the line width to 10.Then trace the longest neurite beginning near the cell body and extending to the tip. Press Control and T then F to add the measurement to the ROI Manager and highlight the measured neuron. Select all numbers in the ROI.Click on Measure, then copy and paste the measurements into a spreadsheet. To measure fluorescence intensity of labeled cells, click on the fourth icon in the toolbar Freehand Selections and draw the shape of the cell. Click Analyze and Set Measurements.Then make sure that Area, Mean gray value, Min max gray value and Integrated density are selected. Click Analyze and measure. Select the region next to the cell as background.And then click analyze and measure. Select all measure data and copy, paste it into a spreadsheet. To coat the plate add 30 microliters of poly-L-lysine into each well of a 384-well plate and incubate the plate at room temperature for one hour.Wash it twice with PBS and let it dry for approximately 30 minutes. Add 30 microliters of laminine to each well, then incubate the plate at 37 degrees Celsius for two hours. Repeat the washes with PBS and proceed with plating the cells.Add 20, 000 single cell neurospheres in 25 microliters of differentiation media to each well of the 384-well plate and incubate the plate at 37 degrees Celsius for five days. Hight precision pipetting skills are required for plating the exact number of neurospheres that's all segregated into single cells. Therefore, differentiating the neurospheres into neurons before plating is recommended to achieve more reproducible results.After the incubation, add five microliters of test compound to each well at six times the desired concentration. And incubate the cells for another 24 hours. To perform the cell viability essay, add 30 microliters of luminescent reagent to each well.And leave the plate on a shaker for two minutes. Centrifuge the plate at 300 to 400 times g for 30 seconds, then incubate the plate at room temperature for 10 minutes, protecting it from light. After the incubation, measure the luminescence on a microplate reader.The human neural progenitor cell-derived neurons were used to examine the effect of HDAC inhibitors as small molecule epigenic compounds for the extension of neurites and subsequent neurogenic ability of small molecule compounds. The neurotoxicity of the HDAC inhibitors was also assessed after differentiating the neural progenitor cells in 384-well plates, showing the potential to scale the protocol for a higher number of compounds. In a different study, measurement of the neuronal cell fluorescence intensity was used to quantify the abundance of histone H4 lysine five acetylation as a histone marker after treating the neurons with epigenetic modifier compounds.Additionally, this protocol has been used to visualize a pre-synaptic protein, synaptophysin, to check for possible synaptogenic effects of small molecule compounds. Using the presented method, neurites number, intensity, length, and width, together with synaptic characteristics, including pre and post-synaptic protein modifications could be reliably measured.

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Research

JoVE Journal

Developmental Biology

7.3K visualizações.  Tarbiat Modares University. O protocolo apresentado descrevendo o ensaio de crescimento de neurite que é altamente relevante para a biologia humana e a avaliação neurotoxicidade de um pequeno composto de moléculas. Alto potencial translacional e relevância fisiológica das células progenitoras neurais humanas como modelo primário de células humanas oferecem uma vantagem considerável na triagem de descoberta de drogas relacionada ao crescimento de neurite e avaliação da neurotoxicidade. Utilizando o protocolo ...