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...

<ol>
	<li>Sherman, S. P., Bang, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screen+for+compounds+that+modulate+neurite+growth+of+human+induced+pluripotent+stem+cell-derived+neurons.">High-throughput screen for compounds that modulate neurite growth of human induced pluripotent stem cell-derived neurons.</a> Disease Models &amp; Mechanisms. 11 (2), (2018).</li><li>Al-Ali, H., Beckerman, S. R., Bixby, J. L., Lemmon, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+axon+regeneration.">In vitro models of axon regeneration.</a> Experimental Neurology. 287, 423-434 (2017).</li><li>Kudo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+neurite+outgrowth+in+PC12+cells+treated+with+temperature-controlled+repeated+thermal+stimulation.">Induction of neurite outgrowth in PC12 cells treated with temperature-controlled repeated thermal stimulation.</a> PloS One. 10 (4), 0124024 (2015).</li><li>Higgins, S., Lee, J. S., Ha, L., Lim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+neurite+outgrowth+by+mechanical+cell+stretch.">Inducing neurite outgrowth by mechanical cell stretch.</a> BioResearch Open Access. 2 (3), 212-216 (2013).</li><li>Muramatsu, R., Ueno, M., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+regenerative+mechanisms+of+central+nervous+system+neurons.">Intrinsic regenerative mechanisms of central nervous system neurons.</a> Bioscience Trends. 3 (5), (2009).</li><li>Read, D. E., Herbert, K. R., Gorman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+enhances+NGF-induced+neurite+elongation+which+is+not+mediated+by+Hsp25+in+PC12+cells.">Heat shock enhances NGF-induced neurite elongation which is not mediated by Hsp25 in PC12 cells.</a> Brain Research. 1221, 14-23 (2008).</li><li>Finan, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+Compound+Screen+for+Pharmacological+Enhancers+of+Apolipoprotein+E+in+Primary+Human+Astrocytes.">Bioactive Compound Screen for Pharmacological Enhancers of Apolipoprotein E in Primary Human Astrocytes.</a> Cell Chemical Biology. 23 (12), 1526-1538 (2016).</li><li>Magistri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+transcriptomic+analysis+of+astrocytes+differentiation+from+human+neural+progenitor+cells.">A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.</a> European Journal of Neuroscience. 44 (10), 2858-2870 (2016).</li><li>Bez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+neurosphere-forming+cells:+morphological+and+ultrastructural+characterization.">Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization.</a> Brain Research. 993 (1-2), 18-29 (2003).</li><li>Grandjean, P., Landrigan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioural+effects+of+developmental+toxicity.">Neurobehavioural effects of developmental toxicity.</a> The Lancet Neurology. 13 (3), 330-338 (2014).</li><li>Finkbeiner, S., Frumkin, M., Kassner, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+screening:+extracting+meaning+from+complex+data.">Cell-based screening: extracting meaning from complex data.</a> Neuron. 86 (1), 160-174 (2015).</li><li>An, W. F., Tolliday, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+assays+for+high-throughput+screening.">Cell-based assays for high-throughput screening.</a> Molecular Biotechnology. 45 (2), 180-186 (2010).</li><li>Astashkina, A., Mann, B., Grainger, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+evaluation+of+in+vitro+cell+culture+models+for+high-throughput+drug+screening+and+toxicity.">A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.</a> Pharmacology &amp; Therapeutics. 134 (1), 82-106 (2012).</li><li>Swinney, D. C., Anthony, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+were+new+medicines+discovered.">How were new medicines discovered.</a> Nature Reviews Drug Discovery. 10 (7), 507 (2011).</li><li>Ryan, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+in+human+induced+pluripotent+stem+cell-derived+neurons+as+a+high-throughput+screen+for+developmental+neurotoxicity+or+neurotoxicity.">Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity.</a> Neurotoxicology. 53, 271-281 (2016).</li><li>Magistri, M., Velmeshev, D., Makhmutova, M., Faghihi, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomics+profiling+of+Alzheimer's+disease+reveal+neurovascular+defects,+altered+amyloid-&#946;+homeostasis,+and+deregulated+expression+of+long+noncoding+RNAs.">Transcriptomics profiling of Alzheimer's disease reveal neurovascular defects, altered amyloid-&#946; homeostasis, and deregulated expression of long noncoding RNAs.</a> Journal of Alzheimer's Disease. 48 (3), 647-665 (2015).</li><li>Darbinyan, A., Kaminski, R., White, M. K., Darbinian, N., Khalili, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+propagation+of+primary+human+and+rodent+embryonic+neural+progenitor+cells+and+cortical+neurons.">Isolation and propagation of primary human and rodent embryonic neural progenitor cells and cortical neurons.</a> Neuronal Cell Culture. , 45-54 (2013).</li><li>Gil-Perot&#237;n, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neural+stem+cells+from+the+subventricular+zone:+a+review+of+the+neurosphere+assay.">Adult neural stem cells from the subventricular zone: a review of the neurosphere assay.</a> The Anatomical Record. 296 (9), 1435-1452 (2013).</li><li>Ebert, A. D., McMillan, E. L., Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating,+expanding,+and+infecting+human+and+rodent+fetal+neural+progenitor+cells.">Isolating, expanding, and infecting human and rodent fetal neural progenitor cells.</a> Current Protocols in Stem Cell Biology. 6 (1), 2 (2008).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676 (2012).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> Biotechniques. 43 (1), 25-30 (2007).</li><li>Bagheri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC+Inhibitors+Induce+BDNF+Expression+and+Promote+Neurite+Outgrowth+in+Human+Neural+Progenitor+Cells-Derived+Neurons.">HDAC Inhibitors Induce BDNF Expression and Promote Neurite Outgrowth in Human Neural Progenitor Cells-Derived Neurons.</a> International Journal of Molecular Sciences. 20 (5), 1109 (2019).</li><li>Sartor, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+BDNF+expression+and+memory+by+HDAC+inhibition+requires+BET+bromodomain+reader+proteins.">Enhancement of BDNF expression and memory by HDAC inhibition requires BET bromodomain reader proteins.</a> Journal of Neuroscience. 39 (4), 612-626 (2019).</li><li>Conde, C., C&#225;ceres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly,+organization+and+dynamics+in+axons+and+dendrites.">Microtubule assembly, organization and dynamics in axons and dendrites.</a> Nature Reviews Neuroscience. 10 (5), 319 (2009).</li><li>Schmitz, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+analysis+of+neuronal+morphology,+synapse+number+and+synaptic+recruitment.">Automated analysis of neuronal morphology, synapse number and synaptic recruitment.</a> Journal of Neuroscience Methods. 195 (2), 185-193 (2011).</li><li>Grandjean, P., Landrigan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neurotoxicity+of+industrial+chemicals.">Developmental neurotoxicity of industrial chemicals.</a> The Lancet. 368 (9553), 2167-2178 (2006).</li><li>Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+human+brain+in+preclinical+drug+development.">The adult human brain in preclinical drug development.</a> Nature reviews Drug Discovery. 7 (8), 659 (2008).</li><li>Dolmetsch, R., Geschwind, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+brain+in+a+dish:+the+promise+of+iPSC-derived+neurons.">The human brain in a dish: the promise of iPSC-derived neurons.</a> Cell. 145 (6), 831-834 (2011).</li><li>Pan, C., Kumar, C., Bohl, S., Klingmueller, U., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomic+phenotyping+of+cell+lines+and+primary+cells+to+assess+preservation+of+cell+type-specific+functions.">Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions.</a> Molecular &amp; Cellular Proteomics. 8 (3), 443-450 (2009).</li><li>Alge, C. S., Hauck, S. M., Priglinger, S. G., Kampik, A., Ueffing, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+profiling+of+primary+versus+immortalized+human+RPE+cells+identifies+expression+patterns+associated+with+cytoskeletal+remodeling+and+cell+survival.">Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival.</a> Journal of Proteome Research. 5 (4), 862-878 (2006).</li><li>Yeo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Embryonic+Stem+Cell-Derived+Neural+Lineages+as+In+Vitro+Models+for+Screening+the+Neuroprotective+Properties+of+Lignosus+rhinocerus+(Cooke)+Ryvarden.">Human Embryonic Stem Cell-Derived Neural Lineages as In Vitro Models for Screening the Neuroprotective Properties of Lignosus rhinocerus (Cooke) Ryvarden.</a> BioMed Research International. 2019, (2019).</li></ol>

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 ...

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.

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

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 ...