The authors thank Priscilla L. Yang and Dominique J. Burri of Harvard Medical School for help with initial propagation and quantification of ZIKV. We also would like to thank Nathaniel D. Kirkpatrick for imaging support. K.E. was supported by the Stanley Center for Psychiatric Research and the Harvard Stem Cell Institute.

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in SCMM medium with a vitronectin or Geltrex substrate. If using an alternative, high-protein stem cell culturing media, it is advisable to transition SC cultures to SCMM for at least two passages before initiating organoid formation. The protocol has not been tested with feeder-based SC culture methods.
<ol>
	<li>Using regular SC maintenance methods25,26, bring cultures to 50%-70% confluence. This is typically 3-4 days after passaging, though this can be dependent on culture method and cell line. 
	Note: Approximately 2 wells from a 6-well plate are needed to produce a full 96-well plate of organoids.</li>
	<li>Inspect cultures via brightfield microscopy at 10X-20X magnification to ensure healthy colony morphology, with no detectable differentiation.</li>
	<li>Bring SCMM to 37 &#176;C in a hot water bath, and thaw a 50 &#181;L vial of 50 mM Y-27632 (Rock inhibitor) and 2 mL of enzymatic detachment reagent to room temperature.</li>
	<li>Prepare an ultra-low attachment (ULA) U-bottom 96 well plate, a multichannel P200 pipette, and a 25 mL reagent reservoir.</li>
	<li>Aliquot 45 mL of SCMM in a 50-mL conical tube, and add 45 &#181;L of 50 mM Y-27632. Mix thoroughly by triturating.</li>
	<li>Vacuum aspirate the two wells containing SCs, and quickly add 1 mL of enzyme-free detachment reagent to each well using a P1000 pipette. Incubate the plate in a 37 &#176;C incubator for 4 min.</li>
	<li>Vacuum aspirate the treated wells, and add 1 mL of enzymatic detachment reagent to each well using a P1000 pipette.
	<ol>
		<li>Incubate the plate in a 37 &#176;C incubator for an additional 5 min.</li>
		<li>Remove the plate and examine the treated wells. Gently tap on the plate to break up aggregated cells. If large cell clusters are still visible, incubate the plate for 2 additional min at 37 &#176;C. Repeat this step until clusters are no longer visible by the naked eye.</li>
	</ol>
	</li>
	<li>Once the cells are properly dissociated, add 1 mL of SCMM to each well to deactivate the dissociation enzymes. Pool the 4 mL of cell suspension in a 50-mL conical tube and take a small aliquot for cell counting.
	<ol>
		<li>Return any remaining, untreated wells in the maintenance plate to the 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>While counting, centrifuge the cell suspension at 300 x g for 5 min.</li>
	<li>Determine the total number of cells, and calculate the amount of SCMM + Y-27632 solution that will be needed to achieve a 60,000 cells/mL suspension.</li>
	<li>Carefully vacuum aspirate the supernatant and add the calculated volume of SCMM + Y-27632 solution. Mix 5-10 times with a 5-mL pipet, ensuring a uniform cell suspension.</li>
	<li>Immediately transfer the cell suspension into a reagent reservoir, and pipette 150 &#181;L into each well of the ULA U-bottom 96-well plate using a multichannel P200 pipette. This produces 96 individual organoids, each initially consisting of 9,000 cells.</li>
	<li>Centrifuge the plate at 150 x g for 1 min.</li>
	<li>Place the plate into a 37 &#176;C incubator, and do not disturb the plate for 48 h.</li>
</ol>
2. Cerebral Organoid Maintenance (Day 2)
<ol>
	<li>Prepare 17 mL of SCMM with 17 &#181;L of 50 mM Y-27643 in a 50-mL conical tube. Warm the SCMM + Y-27632 solution to 37 &#176;C in a hot water bath.</li>
	<li>Take the organoid plate from the incubator, and using a multichannel P200 pipette set to 75 &#181;L, slowly draw up medium from the edge of the well on the first row. Once the medium has been drawn, quickly expel the medium back into the well to lift up loose cells and organoids. Repeat this for each row. 
	Note: This technique, hereafter referred to as a &#34;dispersion,&#34; lifts cellular debris and the organoid into suspension. The organoid rapidly sinks to the bottom of the well, while the smaller debris remains in suspension, allowing it to be removed with a media change. This helps to prevent the organoid from resting in cellular debris during culture.
	<ol>
		<li>After all wells have been dispersed, wait for 15 s to ensure that the organoids have sunk to the bottom of each well.</li>
	</ol>
	</li>
	<li>Slowly and carefully aspirate 75 &#181;L of medium from each well using the multichannel P200 pipette and dispense into a waste reservoir. 
	Note: Most users occasionally aspirate an organoid during regular feeds. This can occur roughly 1% of the time. Please plan accordingly to ensure that enough organoids survive to experimental time points.</li>
	<li>Transfer SCMM + Y-27632 solution into a reagent reservoir, and dispense 150 &#181;L into each organoid well using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
3. Cerebral Organoid Maintenance (Day 3)
<ol>
	<li>Warm 17 mL of SCMM to 37 &#176;C, this time without Y-27643.</li>
	<li>With the multichannel P200 pipette set to 100 &#181;L, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.</li>
	<li>Prepare a waste reservoir and transfer warmed SCMM into a source reservoir.</li>
	<li>Carefully aspirate 125 &#181;L of medium from each well, and replace with 150 &#181;L of fresh SCMM using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
4. Cerebral Organoid Maintenance (Day 4+)
Note: Conduct this regular maintenance every other day until infection.
<ol>
	<li>Before beginning the feed on day 4, prepare Neural Induction (NI) medium.
	<ol>
		<li>Thaw a 250 &#181;L aliquot of 2 mg/mL heparin, along with one 5 mL vial of 100X N-2 Supplement. Add the 250 &#181;L of heparin, 5 mL of 100X N-2, and 5 mL of 100X MEM-NEAA to 500 mL of DMEM/F12+GlutaMAX. Sterile filter the mixed solution in a 500 mL filter. 
		Note: Keep NI at 4 &#176;C when not in use; NI lasts up to two weeks before fresh medium should be prepared.</li>
	</ol>
	</li>
	<li>Warm 17 mL of NI medium to 37 &#176;C.</li>
	<li>With the multichannel P200 pipette set to 100 &#181;L, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.</li>
	<li>Prepare a waste reservoir and transfer warmed NI into a source reservoir.</li>
	<li>Carefully aspirate 125 &#181;L of medium from each well, and replace with 125 &#181;L of fresh NI using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
5. Organoid Infection with ZIKV (~Day 24)
NOTE: After approximately 3 weeks of differentiation, there will be large neuroepithelial structures and rosettes visible within the organoid that will be highly susceptible to ZIKV infection.
<ol>
	<li>Bring Earle&#39;s 1X Balanced Salt Solution (EBSS), 1X PBS, and NI to 37 &#176;C in a hot water bath.</li>
	<li>Dilute ZIKV of known concentration in 1X EBSS to the target infection multiplicity of infection (MOI). 
	Note: Titrations of different MOIs are recommended to achieve a viral dose response. This may vary by virus strain; for ATCC VR-1838 and ATCC VR-1843, typical MOI values range between 0.1 and 10).</li>
	<li>Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 200 &#181;L of 1X PBS to each well using a P200 multichannel pipette to wash the organoids.</li>
	<li>Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 50 &#181;L of 1X EBSS-only (mock infection) or ZIKV virus solution to well and ensure that the organoid is completely submerged. Repeat for each organoid that is to be infected for the study.</li>
	<li>Place the plate back into a 37 &#176;C incubator for 2 h. 
	Note: Incubating mock-infected organoids for this duration should not significantly impact their growth as compared to undisturbed organoids.</li>
	<li>Approximately 30 min before viral incubation is completed, aliquot 25 mL of NI into a 50-mL conical tube, and bring to 37 &#176;C in a hot water bath.</li>
	<li>After viral exposure is completed, wash the organoids with 200 &#181;L of 1X PBS for each well, allow organoids to settle for 15 s, and then remove 1X PBS using a single channel P200 pipette.</li>
	<li>Add 200 &#181;L of fresh NI to each well, and place back into the incubator.
	<ol>
		<li>Optional: For a day 0 time point, organoids may be imaged one hour after infection.</li>
	</ol>
	</li>
	<li>Continue to culture by replacing 125 &#181;L NI every other day using the dispersion method (see step 4). Be sure to properly dispose of spent media and tips, as this contains infectious ZIKV particles.</li>
</ol>

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F., 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=Genetic+Ablation+of+AXL+Does+Not+Protect+Human+Neural+Progenitor+Cells+and+Cerebral+Organoids+from+Zika+Virus+Infection.">Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection.</a> Cell Stem Cell. 19, 703-708 (2016).</li><li>Dang, 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=Zika+Virus+Depletes+Neural+Progenitors+in+Human+Cerebral+Organoids+through+Activation+of+the+Innate+Immune+Receptor+TLR3.">Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.</a> Cell Stem Cell. 19, 258-265 (2016).</li><li>Gabriel, E., 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=Recent+Zika+Virus+Isolates+Induce+Premature+Differentiation+of+Neural+Progenitors+in+Human+Brain+Organoids.">Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.</a> Cell Stem Cell. 20, 397-406 (2017).</li><li>Qian, X., 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=Brain-Region-Specific+Organoids+Using+Mini-bioreactors+for+Modeling+ZIKV+Exposure.">Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.</a> Cell. 165, 1238-1254 (2016).</li><li>Eiraku, 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=Self-Organized+Formation+of+Polarized+Cortical+Tissues+from+ESCs+and+Its+Active+Manipulation+by+Extrinsic+Signals.">Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals.</a> Cell Stem Cell. 3, 519-532 (2008).</li><li>Lancaster, M. 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The authors declare that they have no competing financial interests.

There are several caveats to consider when utilizing human cerebral organoids to investigate ZIKV infection. One important consideration is that organoid formation and structure is highly dependent on the stem cell line from which they are formed. Take care when comparing between cell lines, including isogenic engineered lines; it is best to make conclusions using multiple subclones, and ideally multiple stem cell lines. Additionally due to this difference in cell lines, pilot experiments to ensure that proper differentiation is occurring in the organoids is recommended. While the neuroepithelial structures are visible by brightfield microscopy, it is also suggested to conduct qRT-PCR or cryosectioning experiments to look for neural differentiation markers such as PAX6 or phospho-vimentin.
One should also anticipate considerable variability among organoids within the same cell line. Due to the unpatterned nature of the protocol, the number and size of neuroepithelial structures can vary between individual organoids. Typically, one can expect at least two large (&gt;200 &#181;m in diameter by D24) neural rosettes per organoid. For this reason, longitudinal studies, such as the observation of change in organoid size upon infection, are particularly enlightening. Variability between batches may also occur, with the most likely cause for this being inaccurate cell counting or seeding. It is recommended to conduct multiple counts and to make sure the cell suspension is well mixed before seeding onto the ULA U-bottom 96-well plates. 
When culturing organoids in ULA U-bottom 96-well plates, plan on seeing considerable evaporation effects around the edges of the plates. It is ideal to fill these wells with PBS and only work with the center 60 wells. Due to the evaporation, organoids in the outer wells will be exposed to different conditions than those in the center, which will likely affect their growth and differentiation. Please consider this when planning the number of organoids that will be needed for experiments. Additionally, organoids will begin to overgrow the 96-well format after roughly 30 days, which will be visible due to the discoloration of culture medium. If planning on taking the organoids past the 30 days, it is recommended to transfer the organoids to a ULA 24-well plate. To conduct the transfer, use scissors to cut a P1000 tip so that the opening is much larger than the organoids themselves, and transfer the organoids by pipetting.
Human cerebral organoids hold great potential in advancing the field's understanding of neurodevelopment and disease. Researchers are encouraged to modify the protocol as necessary. For example, one could implement patterning factors to improve differentiation efficiency toward specific cell types, allowing them to explore the viral impacts on certain anatomical regions. The neurodevelopmental effects of ZIKV are still poorly understood, but this new approach will give researchers an accessible, rapid, and high-throughput way of investigating the mechanisms of infection.

Zika virus (ZIKV) has rapidly spread in Micronesia, French Polynesia, and the Americas, and has recently been shown to cross the placental barrier1,2 to infect the developing fetal brain, leading to neurodevelopmental diseases such as microcephaly3,4,5,6 . The long-term impact on these patients&#39; lives, and the current lack of treatment, has led researchers and clinicians to scramble for a better understanding of the mechanisms behind ZIKV infection and replication. Previous studies have examined ZIKV infection of in vitro systems in a variety of physiologically-relevant cell types7,8,9, as well as in vivo10 with immunocompetent and immunocompromised mice11,12,13 and non-human primates14,15,16. In addition to these more conventional techniques, several groups have implemented stem cell-derived cerebral organoids to understand more about ZIKV infection of the developing human brain. These groups have utilized human cerebral organoids to confirm the microcephaly phenotype17,18, investigate receptors linked to virus entry19, examine physiological responses to infection20,21, and potentially screen for drug candidates22. Here a technique for rapidly producing and infecting stem-cell derived cerebral organoids is described, as shown previously19, to improve the understanding of ZIKV infection of the developing human brain.
Since the organoids are formed from standard pluripotent stem cell (PSC) cultures, this technique allows for a variety of scientific questions to be answered regarding viral infection of the developing brain. For example, CRISPR engineering may be used to modify these PSC lines prior to organoid infection at a faster rate than most in vivo genetic studies19. Additionally, unlike in standard two dimensional (2D) differentiation cultures, organoids exhibit the complex cellular architecture that is critical for corticogenesis, and recent studies have shown that this architecture may be disrupted upon infection21.Finally, the relatively low cost of generating organoids allows for higher throughput experimentation and screening when compared to in vivo models. On the other hand, there are some disadvantages to the utilization of organoids to study ZIKV. While organoids are far more biologically relevant than 2D cultures, there are additional challenges in the assessment of organoids due to their three-dimensional (3D) nature. Imaging and dissociation of organoids tends to involve more equipment and a larger investment of resources than in standard 2D cultures. Additionally, organoids lack the vasculature and immunological components that are present in in vivo models, so researchers interested in those aspects of viral infection are advised to seek an alternative protocol.
There are a number of techniques for the formation of human organoids and neurospheres in culture, and they typically fall within the patterned or unpatterned categories. Patterned methods implement factors to regulate Wnt, BMP, TGF&#946;, and other signaling pathways to push differentiation toward specific lineages23. Unpatterned methods, such as the one described here, take advantage of the propensity for induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) to differentiate toward a neuroectodermal lineage by default24. After approximately three weeks of differentiation, the resulting organoids consist of large, biomimetic neuroepithelial structures that contain several cell types that are observed in the early developing brain.
The technique for producing organoids from standard, feeder-free PSC cultures, and infecting these organoids with ZIKV is presented in full. For guidance on the culturing methods needed for feeder-free PSCs, please refer to previous methods publications25,26. Additionally, in order to apply consistent amounts of virus for multiple experiments, it is important to calculate the MOI ahead of time. This is done by conducting an infection of Vero cells, followed by treatment with overlay medium, incubation, and immunostaining. Descriptions and methods of this technique have been previously described19,27.
Once the PSC culture reaches the target confluence of 50%-70%, the cells are then dissociated and aggregated into ultra-low attachment 96-well plates. The cells are maintained for 3 days in xeno-free stem cell maintenance media (SCMM), and then converted to a neural induction media for the remainder of the culture. Once the organoids have differentiated for 3 weeks, the infection can be conducted. By routinely taking images during the week following infection, researchers will observe progressive cell death and disruption of the organoid. Researchers may also dissociate the organoids at this time to conduct transcriptional or proteomic profiling. Cryosectioning and lightsheet methods are recommended for imaging, and researchers can expect to see high levels of infection and viral replication particularly within the neural progenitor cell (NPC) populations in the organoids. Ultimately, this technique allows researchers to rapidly examine the mechanisms of viral infection of the human brain with low cost and limited equipment.

<table><tbody><tr><td>Enzyme-Free Detachment Reagent, ReLeSR</td><td>StemCell Technologies</td><td>5873</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650-100ML</td><td></td></tr><tr><td>Glutamate-supplemented DMEM/F12 (1X)</td><td>Gibco</td><td>10565-018</td><td>(+)2.438 g/L sodium bicarbonate, (+)sodium pyruvate</td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (MEM-NEAA)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)</td><td>GE Healthcare Life Sciences</td><td>SH30029.02</td><td></td></tr><tr><td>Stericup Filter (500mL)</td><td>Millipore</td><td>220009-140</td><td></td></tr><tr><td>Costar Ultra-Low Attachment Plate, 96-Well, Round Bottom</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>Low Residual Volume Reagent Reservoir (25mL)</td><td>Integra</td><td>4312</td><td></td></tr><tr><td>Pipet-Lite Multi Pipette L12-200XLS+</td><td>Rainin</td><td>17013810</td><td></td></tr><tr><td>Large Centrifuge, Culture Plate-Compatible</td><td>Thermo Scientific</td><td>75007210</td><td></td></tr><tr><td>Culture Plate Centrifuge Rotor</td><td>Thermo Scientific</td><td>75005706</td><td></td></tr><tr><td>Vero Cells</td><td>ATCC</td><td>CCL-81</td><td>For virus expansion, if necessary</td></tr><tr><td>Zika Virus (Uganda Strain)</td><td>ATCC</td><td>VR-1838</td><td>Other strains may be used</td></tr><tr><td>Zika Virus (Puerto Rico Strain)</td><td>ATCC</td><td>VR-1843</td><td>Other strains may be used</td></tr><tr><td>phospho-vimentin</td><td>MBL</td><td>D076-3</td><td>mouse polyclonal,&amp;nbsp; 1:250 dilution</td></tr><tr><td>TBR2</td><td>Abcam</td><td>ab23345</td><td>rabbit polyclonal, 1:300 dilution</td></tr><tr><td>MAP2</td><td>Novus</td><td>NB300-213</td><td>chicken polyclonal, 1:1500 dilution</td></tr><tr><td>GFAP</td><td>Cell Signaling Technologies</td><td>CST12389S</td><td>rabbit polyclonal, 1:200 dilution</td></tr><tr><td>D1-4G2 flavivirus envelope protein</td><td>EMD Millipore</td><td>MAB10216</td><td>mouse monoclonal, 1:500 dilution</td></tr><tr><td>cleaved caspase-3</td><td>Cell Signaling Technologies</td><td>9661</td><td>rabbit polyclonal, 1:200 dilution</td></tr><tr><td>488 anti-mouse IgG</td><td>Thermo Fisher</td><td>A-11001</td><td>goat polyclonal, 1:1000</td></tr><tr><td>594 anti-rabbit IgG</td><td>Thermo Fisher</td><td>A-11037</td><td>goat polyclonal, 1:1000 dilution</td></tr><tr><td>647 anti-chicken IgY</td><td>Thermo Fisher</td><td>A-21449</td><td>goat polyclonal, 1:10&lt;span class=&quot;font0&quot;&gt;00 dilution&lt;/span&gt;</td></tr></tbody></table>

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.

During the day of organoid formation, SC cultures should be in the log-growth phase, in which they are 50%-70% confluent, as shown in Figure 1(A-C). Cultures should form rounded colonies with distinct edges, and the wells should be clear of differentiation Figure 1(D-E). If differentiation is occurring, it is recommended to conduct a pick-to-remove to clean the affected areas within the plates at least one day prior to creating organoids. If a large portion of the culture is differentiated, it may be necessary to conduct an extra passage, thaw a new vial, or alter stem cell culture techniques to ensure that the organoids are being formed by pure stem cell cultures.
The dissociation by a combination of enzyme-free and enzymatic detachment reagents treatment should lead to mostly single cells, though there may still be small aggregates of cells observed during counting. The presence of small aggregates does not appear to disrupt organoid formation, though it may affect counting accuracy. It is recommended that multiple counts are made per sample, and if the counts vary by over 20%, it may be necessary to increase dissociation time to singularize cells further.
Once the cells are seeded and spun down in a ULA U-bottom 96-well plate (Figure 2A), they will appear as a flat, dense sheet of cells on the bottom of each well. The cells will slowly aggregate over the next two days. It is best not to disturb the plate during these 48 hours, as mechanical forces may disrupt the loosely-formed aggregate. For the first 10 days, the organoid will remain mostly spherical, and will grow from ~300 &#181;m to 600-800 &#181;m (Figure 2B). More observable structures begin to appear in the organoid between day 10 and day 20(Figure 2C-E). By day 24, researchers should be able to observe rosette-like structures via brightfield microscopy (Figure 2F). While continuing the culture, these neuroepithelial structures may increase in quantity and size over several days (Figure 2G). During feeds, the researcher will notice considerable amounts of debris collecting on the bottom of each well, particularly during the first 2 weeks of culture. The dispersion technique described in step 2.2 removes most of this cellular debris to prevent the apoptotic debris from accumulating over multiple feeds (Figure 3A). This debris can interfere with imaging, it can make it more challenging to observe and quantify infection-induced cell death, and it may provide asymmetric signaling to the neighboring organoid that could affect differentiation. If the dispersion technique is properly conducted, this should be reduced (Figure 3B-C, 3D-E).
It is recommended to conduct cryosections or lightsheet imaging to inspect the cortical structure forming within the organoids. In day 25 organoids, the rosette structures and ventricular zones are clearly visible in DAPI (Figure 4A) and phospho-vimentin (Figure 4B) staining. The presence of TBR2 (Figure 4C) indicates that intermediate progenitors are forming, with a morphology that suggests outward migration. MAP2+ (Figure 4D) neurons populate the outer regions of each rosette. From these proteins, one can visualize the ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP) within each rosette (Figure 4E,4E&#39;). One can continue to culture these organoids in order to observe later stages of development. While cortical layering is not as prominent in this type of organoid, the natural switch to gliogenesis does occur much like it does in vivo. This can be observed in day 108 organoids, in which a large portion of the cells express either MAP2 or GFAP (Figure 4F-I).
The users may expect to see differences in organoid size by approximately 3 days after ZIKV infection, depending on the viral MOI applied to the organoids (Figure 5A-B). After these 3 days, there will also be an increase in cellular debris in the infected wells compared to the mock wells. This difference in organoid size will increase over the following week, until the infected organoids begin to break apart (Figure 5C-D). A variety of assays may be used at this point to investigate infection mechanisms, including viral RNA extraction and cryosectioning (recommended antibodies reported in the Table of Materials). Upon cryosectioning, users will observe a large presence of virus in the apical region of neuroepithelial structures, suggesting a susceptibility of neural progenitor cells (NPCs) to ZIKV infection (Figure 6). Immunofluorescence of sectioned organoids will also show an increase in cleaved caspase-3 expression in the infected organoids (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56404/56404fig1.jpg" /> 
Figure 1: Stem cell colony morphology prior to organoid formation. 
Colonies should be 50%-70% confluent at the time of dissociation to produce a large number of consistent organoids. (A) Sparse, recently-seeded cultures have not yet reached log-growth phase, and will not produce many organoids. (B) Once the cells reach the proper confluence, they are ready for dissociation. It is also important that these colonies are inspected to ensure that they are clear of differentiation. (C) Colonies that are taken too far will reach over-confluence and are not recommended for organoid formation. Cultures in this state are more likely to contain differentiating cells. (D) Colony edges should be distinct, with consistent cell morphology of rounded cells in the center, and slightly elongated cells toward the edges. (E) Minimal differentiation should be present in the cultures. If larger, flattened cells are observed in the centers or edges of colonies (white arrowheads) and make up more than roughly 1% of the culture, it is recommended that pick-to-remove or additional passages are conducted to form a uniform stem cell population.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig1large.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/56404/56404fig2.jpg" /> 
Figure 2: Cerebral organoid growth over time.&#160; 
(A) A diagram for the production of cerebral organoids from 2D PSCs maintained in xeno-free, feeder-free medium. (B) For the first 8 days, organoids will remain mostly spherical with few detectable features. The diameter of the organoid will range from approximately 300 &#181;m to 500 &#181;m. (C-E) At day 10, clearer regions will start to be detectable around the periphery of the organoids, and they will lose their spherical shape. They will continue to increase in size to approximately 1 mm in diameter, depending largely on the cell line being used to produce the organoids. (F) After approximately 20 days of differentiation, users will observe the formation of neuroepithelial structures (black arrowheads) in the clear peripheral regions of most organoids. These have a ring-like morphology, with apical and basal structures mimicking those of the developing cortex. (G) These neuroepithelial structures will continue to grow and increase in size for approximately 20-30 days.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig2large.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/56404/56404fig3.jpg" /> 
Figure 3: Clearing of cellular debris via dispersion feed technique. 
During organoid growth, a large amount of cell death is to be expected, and can lead to accumulation of cellular debris surrounding the base of the organoid. (A) The depicted dispersion technique allows researchers to remove most of this debris during each feed. To do this, using a multichannel P200 pipette, slowly draw up the used medium and rapidly expel it to lift the organoid and cell debris into suspension. The organoid will quickly drop to the bottom of the well, at which time a regular feed can be conducted. Examples of a day 6 organoid before (B) and after (C) dispersion feeding shows considerable debris reduction. This continues for several weeks, as shown by a day 18 organoid before (D) and after (E) dispersion feeding.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig3large.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/56404/56404fig4.jpg" /> 
Figure 4: Characterization of cerebral organoids via immunohistochemistry. 
Lightsheet images of cerebral organoids are shown to provide an example of the cellular architecture that forms. At day 25, DAPI (A) and phosphor-Vimentin (B) indicate individual rosettes and the ventricular zones, respectively. (C) TBR2 labels intermediate progenitors that are migrating outward, and MAP2 (D) labels neurons that have formed in the cortical plate. (E,E&#39;) The combination of these proteins clearly shows the recapitulation of cortical development in the cerebral organoid models. (F-I) By day 108, gliogenesis has occurred, with a mixture of GFAP+ and MAP2+ cells populating much of the organoid. VZ = ventricular zone, SVZ = subventricular zone, IZ = intermediate zone, CP = cortical plate.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56404/56404fig5.jpg" /> 
Figure 5: Degradation of infected organoids.&#160; 
(A) Bright field images of Mock and ZIKV-infected cerebral organoids at MOI = 0.1 and 10. Images were taken immediately after infection (Day 0), as well as 3 and 6 days post-infection. (B-C) Brightfield images of the cell debris surrounding (B) mock and (C) ZIKV-infected at MOI = 10 cerebral organoids 3 days post-infection.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56404/56404fig6.jpg" /> 
Figure 6: Cryosection and immunofluorescence of infected organoids. 
Day 24 organoids were exposed to ZIKV at MOI = 0.1, and cryosectioned 6 days later. At this stage, there is a clear presence of virus in the ventricular, subventricular, and intermediate zones of the organoid, where neural progenitor cells and radial glia reside. (A) Via nuclear staining, these neuroepithelial regions are clearly visible due (white arrows) to the high density of nuclei that form ring-like structures around the apical surface. (B) By staining for the viral envelope, one can observe a relatively high presence of virus within these rosette structures (white arrowheads). Note that there is a small amount of virus present in various unidentified peripheral cells. (C-D) MAP2 or other neuron-specific stains show that there are neurons present in the cultures that do not appear to be infected by the virus when overlaid with the viral stain.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56404/56404fig7.jpg" /> 
Figure 7: Cleaved caspase-3 immunofluorescence of infected organoid. 
After infection, one may use apoptotic indicators to investigate the mechanisms of cell death that occur within the organoids. In this example, day 24 organoids were exposed to ZIKV at MOI=0.1, and cryosectioned 6 days later. (A) Uninfected organoids show no viral envelope (4G2 protein), and a minimal amount of cleaved caspase-3 due to homeostatic apoptosis occurring in the tissue. (B) Infected rosettes begin to show increased levels of apoptosis. (C) Rosettes that are showing severe infection via 4G2 staining also tend to exhibit high levels of cleaved caspase-3 in these regions. There is a direct correlation between infection severity and cleaved caspase-3 expression within the infected rosettes.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56404/56404fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids at JoVE.com

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.

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 ...

modelling-zika-virus-infection-developing-human-brain-vitro-using

Research

JoVE Journal

Neuroscience

10.4K Views.  Novartis Institutes for BioMedical Research. Dit protocol beschrijft een techniek gebruikt om te modelleren Zika virusinfectie van de ontwikkeling van de menselijke hersenen. Wildtype of gemanipuleerde stamcellijnen kunnen, onderzoekers gebruiken deze techniek te ontdekken van de verschillende mechanismen of behandelingen die vroege infectie van de hersenen en de resulterende microcefalie in Zika virus-geïnfecteerde embryo's kunnen beïnvloeden.

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

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Developmental Biology

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.

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 ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

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

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

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 ...

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

Source: Salick, M. R., et al. <a href="https://app.jove.com/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.

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

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

Neuronal Culture Techniques

Advanced Neuronal Models

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

Source: Herrlinger, S. A. et al. <a href="https://app.jove.com/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.

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