Name: zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis
Uploaded: 2018-02-05T14:30:00
Description: Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

This work was supported by funds from the John S. Dunn Foundation and the Institute for Human Infections and Immunity of the University of Texas Medical Branch (P.W.).

Human neural stem cells were originally derived from discarded human fetal cortexes in the first trimester12. All protocol procedures adhere to the University of Texas Medical Branch ethics guidelines concerning the use of human tissue samples, and the cell lines were approved by the Institutional Biosafety Committee.
1. Stock medium preparation and stem cell recovery
<ol>
	<li>Prepare culture medium stock (DFHGPS) by combining the reagents in steps 1.1.1.-1.1.5.
	<ol...

<ol>
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B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Wu, P., 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=Region-specific+generation+of+cholinergic+neurons+from+fetal+human+neural+stem+cells+grafted+in+adult+rat.">Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat.</a> Nat Neurosci. 5 (12), 1271-1278 (2002).</li><li>McGrath, E. 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The ability to culture and manipulate hNSCs provides a critical tool that can be used for a variety of purposes from modeling human disease to high throughput drug screening10,11,12,14,15,16,17. Many questions remained to be addressed such as how human fetal brain NSCs or their progeny are s...

Zika virus (ZIKV) is a flavivirus transmitted either sexually, or by the mosquito vectors Aedes aegypti and Aedes albopictus mosquitoes. ZIKV was recently identified as a severe public health threat due to its ease of transmission and affiliated neurological symptoms1. One of the most concerning neurological effects is the development of microcephaly in fetuses born to pregnant infected mothers2,3. Microcephaly is a neurodevelopmental disorder where the head is smaller than the typical size during fetal development and at birth, with the circumference less than 2 standard deviations below the mean4. The smaller head circumference is commonly accompanied by a variety of comorbidities such as developmental delays, seizures, vision and hearing loss, and feeding difficulty.
Recent studies have used animal models or induced pluripotent stem cells to study the effect of ZIKV infection on neurodevelopment5,6,7,8. While these studies have contributed to our knowledge of ZIKV, the use of different species or genetically modified cells may be time consuming and/or add additional variables that may confound the effect of ZIKV on developing neural cells5,6,7,8. However, the difficulty with hNSC culture, particularly the non-adherent neurosphere culture described in this protocol, is that the culture is very sensitive to the methods used to conduct the culture9. Any change in medium components, or even the physical handling of the culture vessel, is enough to elicit a reaction from the cells9. To address these issues, we developed an in vitro human fetal brain-derived neural stem cell (hNSCs) culture to mechanistically interrogate the effect of ZIKV on fetal neural stem cells. Using our method, hNSCs were maintained for over 80 passages without apparent phenotypic changes10. Furthermore, chromosomal alterations such as trisomy were either none or minimal11. This hNSC culture grows as a non-adherent neurosphere culture. One advantage of neurospheres is that the spheres create a unique environmental niche in culture that is more reflective of in vivo niches compared to two-dimensional cultures9. Another advantage of this protocol is that multiple cell types can be derived from the hNSC culture, enabling an investigator to observe the impact of a given variable on hNSC survival and differentiation. This protocol is applicable to individuals looking to answer mechanistic questions regarding central nervous system development or dysfunction. The following protocol describes how to expand a hNSC culture to infect with ZIKV, and subsequently differentiate the hNSCs to observe the impact of infection on the differentiation process. It also includes methods to store hNSCs for long-term usage, and to differentiate hNSCs into various types of neurons that allow further investigation of ZIKV-induced deficits contributing to brain malformation11. We believe this protocol is also of interest to investigators seeking to understand the impact of any environmental stimulus such as infection or toxins on neural stem cell survival and differentiation.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (10%)</td>
			<td>Sigma</td>
			<td>G8644</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Corning/cellgro</td>
			<td>25-060-c1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep (100x)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I4011</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sigma</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fibroblast growth factor</td>
			<td>R&#38;D system</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>R&#38;D system</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>leukemia inhibitory factor</td>
			<td>R&#38;D system</td>
			<td>7734-LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine hydrobromide(PDL)</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning/cellgro</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4378-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson ImmunoResearch Lab</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>FisherBiotech</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>611659</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Class III beta-tubulin antibody (TuJ1)</td>
			<td>Covnce</td>
			<td>MMS-435p</td>
			<td>1:2000 dulution</td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Millipore</td>
			<td>AB5804</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>ZIKV antibody</td>
			<td>World Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical Branch</td>
			<td></td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Fluoromount G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Forma</td>
			<td>Model# 3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher Scientific</td>
			<td>75004221</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Forma scientific Claas II A/B3</td>
			<td>Model# 1284</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C)</td>
			<td>Forma scientific,Inc</td>
			<td>model# 8516</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope(phase contrast image)</td>
			<td>Hp 2230 workstation</td>
			<td>Product#NOE25us#ABA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (epifluorescent image)</td>
			<td>Nikon Eclipse 80i</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon TE2000-E microscope with C1si confocal system</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

Cultured hNSCs in their proliferative stage will grow as non-adherent neurospheres (Figure 1). Immediately following hNSC passage, there will be many individual cells, which will aggregate and begin to form spheres in the next few days (Figure 1A and 1B). Healthy spheres should be approximately 1-2 mm in diameter 9-10 days following a passage (Figure 1C). Spheres that grow larger tha...

Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

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