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.).

<ol>
	<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All countries and territories with active Zika virus transmisson. , (2016).</li><li>Brasil, 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=Zika+Virus+Infection+in+Pregnant+Women+in+Rio+de+Janeiro.">Zika Virus Infection in Pregnant Women in Rio de Janeiro.</a> N Engl J Med. 375 (24), 2321-2334 (2016).</li><li>Hills, S. L., 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=Transmission+of+Zika+Virus+Through+Sexual+Contact+with+Travelers+to+Areas+of+Ongoing+Transmission+-+Continental+United+States,+2016.">Transmission of Zika Virus Through Sexual Contact with Travelers to Areas of Ongoing Transmission - Continental United States, 2016.</a> MMWR Morb Mortal Wkly Rep. 65 (8), 215-216 (2016).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Facts about microcephaly. , (2016).</li><li>Garcez, P. 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=Zika+virus+impairs+growth+in+human+neurospheres+and+brain+organoids.">Zika virus impairs growth in human neurospheres and brain organoids.</a> Science. 352 (6287), 816-818 (2016).</li><li>Li, 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=Zika+Virus+Disrupts+Neural+Progenitor+Development+and+Leads+to+Microcephaly+in+Mice.">Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice.</a> Cell Stem Cell. 19 (5), 672 (2016).</li><li>Tang, H., 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=Zika+Virus+Infects+Human+Cortical+Neural+Progenitors+and+Attenuates+Their+Growth.">Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth.</a> Cell Stem Cell. 18 (5), 587-590 (2016).</li><li>Wu, K. 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=Vertical+transmission+of+Zika+virus+targeting+the+radial+glial+cells+affects+cortex+development+of+offspring+mice.">Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice.</a> Cell Res. 26 (6), 645-654 (2016).</li><li>Jensen, J. 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. L., 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=Differential+Responses+of+Human+Fetal+Brain+Neural+Stem+Cells+to+Zika+Virus+Infection.">Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection.</a> Stem Cell Reports. 8 (3), 715-727 (2017).</li><li>Svendsen, C. N., 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+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85 (2), 141-152 (1998).</li><li>Tarasenko, Y. I., Yu, Y., Jordan, P. M., Bottenstein, J., Wu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+growth+factors+on+proliferation+and+phenotypic+differentiation+of+human+fetal+neural+stem+cells.">Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells.</a> J Neurosci Res. 78 (5), 625-636 (2004).</li><li>Barrows, N. 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=A+Screen+of+FDA-Approved+Drugs+for+Inhibitors+of+Zika+Virus+Infection.">A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.</a> Cell Host Microbe. 20 (2), 259-270 (2016).</li><li>Jakel, R. J., Schneider, B. 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=Using+human+neural+stem+cells+to+model+neurological+disease.">Using human neural stem cells to model neurological disease.</a> Nat Rev Genet. 5 (2), 136-144 (2004).</li><li>Lopez-Garcia, I., 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=Development+of+a+stretch-induced+neurotrauma+model+for+medium-throughput+screening+in+vitro:+identification+of+rifampicin+as+a+neuroprotectant.">Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant.</a> Br J Pharmacol. , (2016).</li><li>Mich, J. 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=Prospective+identification+of+functionally+distinct+stem+cells+and+neurosphere-initiating+cells+in+adult+mouse+forebrain.">Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.</a> Elife. 3, e02669 (2014).</li></ol>

The authors have no conflicts of interest to declare.

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 susceptible to ZIKV infection, whether different strains of ZIKV infect NSCs with equal efficiency, and how infection during neural development results in microcephaly. We have used this hNSC culture to investigate Zika virus associated neuropathology11,14. By using three strains of cells isolated from three individual donors, we are also able to compare individual differences to susceptibility to the neurological deficits observed following ZIKV infection11. One of the limitations of this technique is the accessibility to hNSC samples and the conditioned medium essential for proper culture. To circumvent this barrier, please contact the corresponding author to arrange a material transfer as the cells used in this protocol are not commercially available.
In vitro studies using hNSCs not only provide unique insight into basic stem cell behavior, they also provide an excellent platform to conduct pre-clinical research. However, the technical challenges of culturing hNSCs can serve as an obstacle in using them as effective and reproducible models. In this protocol, we have outlined key steps and methodologies that can be used to reduce variability in culturing methods and yield a healthy and stable hNSC stem cell culture. A drawback of using hNSC neurosphere culture is that the cells are extremely sensitive to any and all stimuli. Even with the detailed protocol above, it is critical to pay close attention to how much the flasks or dishes of hNSCs are handled, as the subtlest variations on the protocol can result in changes in the neurosphere behavior.
The most crucial part of this methods paper is the cocktail of growth factors and preparation of the different media. If the doubling time of the hNSC culture is very low, or many cells adherie to the culture vessel (when they should be non-adherent), the first step is to ensure that the growth factors being used are the appropriate concentration and not expired. All growth factors in this protocol have a very short shelf life once thawed (5-6 days). Additionally, we have used growth factors from multiple companies and noticed differences in the quality and concentration needed to maintain the culture. Therefore, we highly recommend using the exact growth factors detailed in the Materials Table. The passaging step (steps 3.1-3.22) is also a common source of error. If there are many dead cells following the dissociation process, reduce the number of times of pipetting up and down to dissociate the cells, as too much physical dissociation can result in cell death. Alternatively, if there are many clusters of cells following the dissociation step, increase the vigor or number of times of pipetting up and down, as these clusters will quickly become large spheres that will not remain viable in culture. If the culture is properly maintained, infection with ZIKV is relatively straightforward.
While ZIKV infection is the treatment detailed in this protocol, it is possible to use this hNSC culture system for a wide array of studies. This model system can be used to screen drugs, examine individual susceptibility, and investigate underlying mechanisms of a variety of diseases and developmental disorders14. This system is also ideal for genomic studies since the cultured cells have not been genetically manipulated and show little to no chromosomal abnormalities, even up to 80 passages10,11. We believe this culture system is ideal for modeling in vitro how environmental stimuli such as infection, drugs, alcohol, toxins, etc. can influence development of the nervous system.

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&amp;amp;D system</td><td>233-FB</td><td></td></tr><tr><td>epidermal growth factor</td><td>R&amp;amp;D system</td><td>236-EG</td><td></td></tr><tr><td>leukemia inhibitory factor</td><td>R&amp;amp;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&#039;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>CO&lt;sub&gt;2&lt;/sub&gt; 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 &amp;deg;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 than 2 mm will develop a dark center and the growth factors in the medium will not be able to reach the cells in the center of the sphere, resulting in unwanted differentiation. Healthy spheres will appear translucent and display pseudo-cilia around the edges of the sphere (Figure 1C).
Plating neurospheres on an adherent surface for staining purposes will result in the growth of adherent spheres (Figure 2A). These spheres will touch the surface of the culture vessel and have some projections stretching out on the surface of the vessel, while maintaining a central sphere (Figure 2A). Priming and differentiating the cells require the cells to adhere to a cover slip at the bottom of a cell culture plate, such as a 24-well plate. Following the appropriate priming steps and addition of differentiation medium, the cells will spread out across the surface of the culture vessel and grow as interconnected monolayer of cells (Figure 2B).
To either confirm the validity of the hNSC culture or phenotype of differentiated cells, fluorescent immunohistochemistry can be used. Nestin is an intermediate filament commonly expressed in NSCs and can be used to verify hNSC culture (Figure 3A). To verify the presence of neurons following differentiation, class III &#946;-tubulin can generally be used to label neurons (Figure 3B). Although the priming step is used to achieve a higher percentage of neurons following differentiation, there will still be astrocytes in the culture. Glial fibrillary acidic protein (GFAP) can be used to label astrocytes in order to quantify which percentage of differentiated cells became astrocytes or neurons.
We found that the K048 line of NSCs was infected by both African and Asian strains of ZIKV detected by immunofluorescent staining with a specific ZIKV antibody. The representative images are shown in Figure 4. Mock infection did not elicit a change in hNSC morphology or survival (Figure 4A, 4C). ZIKV tends to stay in the peripheral region of an infected cell one day post a 1-hour infection, but fills the whole cell at 3 to 7 days (Figure 4B, 4D, 4E). Noticeably, with the same MOI (0.1), an estimated 5% infection rate in K048 cells is substantially lower than those recently reported ZIKV infection rate (up to 80%) in human skin-induced NSCs7. This study, reporting 80% infectivity, used the prototype African strain of ZIKV (MR766) that may have adapted a neurotropic phenotype during over 140 passages in mouse brain cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56917/56917fig1.jpg" /> 
Figure 1: Bright field images of hNSC culture. (A-C) Representative bright field images taken at 10X magnification of hNSC culture 1, 4 and 9 days after passaging, respectively. Scale bars: 60 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56917/56917fig2.jpg" /> 
Figure 2: Bright field images of adherent cultures. (A) Representative bright field images taken at 10X magnification of adherent hNSC culture, 7 days after plating. (B) Representative bright field images taken at 10X magnification of adherent differentiated cells following ELL priming and 9 days of differentiation. Scale bars: 60 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56917/56917fig3.jpg" /> 
Figure 3: Fluorescent images of hNSCs and differentiated cultures. (A) Representative fluorescent images taken at 20X magnification of adherent hNSCs stained with nuclear marker, DAPI (blue), and stem cell marker Nestin (red). Scale bar: 60 &#181;m (B) Representative fluorescent images taken at 60X magnification of differentiated cells stained with neural marker &#946;III-tubulin (green), astrocyte maker GFAP (red), and nuclear marker DAPI (blue). Scale bars: 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56917/56917fig4.jpg" /> 
Figure 4: ZIKV infection in human fetal brain-derived NSCs. Human NSCs were either Mock treated (A and C), inoculated for 1 hour with an African lineage strain of ZIKV at MOI of 0.1 (B and D), or with an Asian strain of ZIKV recently isolated from mosquitoes in Mexico in 2016 (E). Immunofluorescent staining detects labeling of ZIKV antibodies in NSCs at one to seven days post inoculation (1 dpi in B, 7dpi in D, and 3 dpi in E). Scale bars 10 &#181;m in A-D, and 5 &#181;m in E. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56917/56917fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFHGPS media for one T75&#160;</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>TPPS*</td>
			<td>173 &#181;L</td>
		</tr>
		<tr>
			<td>200 mM L-Glutamin</td>
			<td>50 &#181;L</td>
		</tr>
		<tr>
			<td>10 mg/mL Insulin**</td>
			<td>25 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL Epidermal growth factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL Basic fibroblast growth factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>10 &#181;g/mL Leukemia inhibitor factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>5 mg/mL Heparin</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td colspan="2">* Mixture containing 100 &#181;g/mL Transferrin, 100 &#181;M putresine, 20 nM progesterone, and 30 nM sodium selenite.&#160; The mixture is made from concentrated stocks, and aliquots are stored at -80 &#176;C and preferablly used within 6 weeks preparation.&#160;&#160; The concentrated stocks are 10 mg/mL transferrin, 30 mM putrescine, 10 &#181;M progesterone and 15 &#181;M sodium selenite stored at -80 &#730;C.</td>
		</tr>
		<tr>
			<td colspan="2">** Insulin is dissolved in 0.01 N hydroen chloride, filtered through&#160; 0.2 &#181;M low protein binding filter, and stored at 4 &#730;C for up to 6 weeks.</td>
		</tr>
	</tbody>
</table>
Table 1: Growth factors for New Proliferation Medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>dPBS*</td>
			<td>1 mLa</td>
			<td>3 mLb</td>
		</tr>
		<tr>
			<td>10% glucose</td>
			<td>60 &#181;L</td>
			<td>180 &#181;L</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>10 &#181;L</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>Dnase**</td>
			<td>5 &#181;L</td>
			<td>15 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">* Dulbecco&#39;s phosphate-buffered saline</td>
		</tr>
		<tr>
			<td colspan="2">** Deoxyribonuclease</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">a for 10 million cells</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">b for 30 million cells</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Preparation of trypsin.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Conditioned media</td>
			<td>1 mLa</td>
			<td>3 mLb</td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor</td>
			<td>10 &#181;L</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>a for 10 million cells</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>b for 30 million cells</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Preparation of trypsin inhibitor.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFHGPS*</td>
			<td>0.7 mLa</td>
			<td>2.1 mLb</td>
		</tr>
		<tr>
			<td>FBS** 20%</td>
			<td>0.2 mL</td>
			<td>0.6 mL</td>
		</tr>
		<tr>
			<td>DMSO*** 10%</td>
			<td>0.1 mL</td>
			<td>0.3 mL</td>
		</tr>
		<tr>
			<td colspan="3">* DMEM, F12, HEPES, glucose, penicillin-streptomycin</td>
		</tr>
		<tr>
			<td colspan="2">** Fetal bovine serum</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">*** Dimethyl sulfoxide</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">a for 5 million cells in one vial</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">b for three vials, 5 million cells/vial</td>
		</tr>
	</tbody>
</table>
Table 4: Preparation of freezing medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFGHPS* for one well in a 24-well plate</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>TPPS**</td>
			<td>17.3 &#181;L</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>10 mg/mL Insulin</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL Epidermal growth factor</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>10 &#181;g/mL Leukemia inhibitory factor</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>1 mg/mL Laminin</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td colspan="2">* Containing DMEM, F12, HEPES, glucose, penicillin-streptomycin</td>
		</tr>
		<tr>
			<td colspan="2">** Containing 100 &#181;g/mL Transferrin, 100 &#181;M putresine,</td>
		</tr>
		<tr>
			<td colspan="2">20 nM progesterone, and 30 nM sodium selenite</td>
		</tr>
	</tbody>
</table>
Table 5: Preparation of ELL priming medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFGHPS* for one well in a 24-well plate</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>TPPS**</td>
			<td>17.3 &#181;L</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>10 mg/mL Insulin</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL basic fibroblast growth factor</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>5 mg/mL Heparin</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>1 mg/mL Laminin</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td colspan="2">* Containing DMEM, F12, HEPES, glucose, penicillin-streptomycin</td>
		</tr>
		<tr>
			<td colspan="2">** Containing 100 &#181;g/mL Transferrin, 100 &#181;M putresine,</td>
		</tr>
		<tr>
			<td colspan="2">20 nM progesterone, and 30 nM sodium selenite</td>
		</tr>
	</tbody>
</table>
Table 6: Preparation of FHL priming medium.

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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Nanyang Technological University

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Developmental Biology

6.4K Views.  University of Texas Medical Branch. 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.

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Immunology and Infection

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Neuroscience

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Source: Salick, M. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

Opracowanie modelu in vitro zakażenia wirusem Zika w organoidach mózgowych

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

JoVE Encyclopedia of Experiments

Neuropathology

Infectious Diseases

Zika Virus Infection of Fetal Neural Stem Cells

Source: McGrath, E. L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
...

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Source: Herrlinger, S. A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

Opracowanie modelu neurodegeneracji wywołanej wirusem Zika u dorosłej myszy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

Source: McGrath, E. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018).
This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor cells.

Różnicowanie ludzkich nerwowych komórek macierzystych w progenitory neuronów i astrocytów

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem ...

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

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Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

Opracowanie modelu in vitro zakażenia wirusem Zika w organoidach mózgowych

Zika Virus Infection of Fetal Neural Stem Cells

Opracowanie modelu neurodegeneracji wywołanej wirusem Zika u dorosłej myszy

Różnicowanie ludzkich nerwowych komórek macierzystych w progenitory neuronów i astrocytów