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Short Article

Zika Virus Depletes Neural Progenitors in Human


Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3
Graphical Abstract Authors
Jason Dang, Shashi Kant Tiwari,
Gianluigi Lichinchi, Yue Qin,
Veena S. Patil, Alexey M. Eroshkin,
Tariq M. Rana

Correspondence
trana@ucsd.edu

In Brief
Dang et al. show that Zika virus (ZIKV)
attenuates growth in cerebral organoids
from human embryonic stem cells by
targeting neural progenitors. ZIKV
activates the TLR3-mediated innate
immune response, leading to
dysregulation of a network of genes
involved in neurogenesis, axon guidance,
apoptosis, and differentiation.

Highlights Accession Numbers


d hESC-derived cerebral organoids model fetal brain GSE80264
development

d Zika virus infects neural progenitor cells in organoid and


neurosphere models

d Zika virus activates Toll-like receptor 3 in cerebral organoids

d TLR3 triggers apoptosis and attenuates neurogenesis

Dang et al., 2016, Cell Stem Cell 19, 18


July 7, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.stem.2016.04.014
Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

Cell Stem Cell

Short Article

Zika Virus Depletes Neural Progenitors in Human


Cerebral Organoids through Activation
of the Innate Immune Receptor TLR3
Jason Dang,1,2,6 Shashi Kant Tiwari,1,6 Gianluigi Lichinchi,1,3 Yue Qin,1 Veena S. Patil,1 Alexey M. Eroshkin,4
and Tariq M. Rana1,5,*
1Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
2Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA
3Graduate School of Biomedical Sciences
4Bioinformatics core

Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
5Institute for Genomic Medicine, University of California San Diego, La Jolla, CA 92093, USA
6Co-first author

*Correspondence: trana@ucsd.edu
http://dx.doi.org/10.1016/j.stem.2016.04.014

SUMMARY outbreak of ZIKV in Brazil and throughout Latin America, new


data suggest a positive correlation between cases of infection
Emerging evidence from the current outbreak of Zika and the rise of microcephaly, characterized by abnormally small
virus (ZIKV) indicates a strong causal link between brains (Driggers et al., 2016; Mlakar et al., 2016; Petersen et al.,
Zika and microcephaly. To investigate how ZIKV 2016). In fact, ZIKV was detected by electron microscopy and
infection leads to microcephaly, we used human RT-qPCR in brains and amniotic fluid of microcephalic fetuses,
embryonic stem cell-derived cerebral organoids to strengthening the causal link between ZIKV and increased inci-
dence of microcephaly (Calvet et al., 2016; Mlakar et al., 2016).
recapitulate early stage, first trimester fetal brain
Furthermore, recent studies show that ZIKV can infect
development. Here we show that a prototype strain
human iPSC-derived neural progenitor cells (NPCs) in vitro, result-
of ZIKV, MR766, efficiently infects organoids and ing in dysregulation of cell-cycle-related pathways and increased
causes a decrease in overall organoid size that corre- cell death (Tang et al., 2016). Evidence thus far suggests a strong
lates with the kinetics of viral copy number. The innate causal relationship between ZIKV and microcephaly.
immune receptor Toll-like-Receptor 3 (TLR3) was up- To investigate the mechanisms by which ZIKV induces micro-
regulated after ZIKV infection of human organoids and cephaly and other neurological disorders, it is essential to use
mouse neurospheres and TLR3 inhibition reduced the scalable, reproducible in vitro models capable of recapitulating
phenotypic effects of ZIKV infection. Pathway anal- complex neurodevelopmental events during early embryogen-
ysis of gene expression changes during TLR3 activa- esis. Advances in embryonic stem cell (ESC) and induced
tion highlighted 41 genes also related to neuronal pluripotent stem cell (iPSC) technology have opened up new
avenues of disease modeling in vitro (Yamanaka, 2012). To
development, suggesting a mechanistic connection
study neurodegenerative disorders, ESCs and human-derived
to disrupted neurogenesis. Together, therefore, our
iPSCs can be differentiated toward forebrain-, midbrain-, and
findings identify a link between ZIKV-mediated TLR3 hindbrain-specific neuron subtypes to model various regions
activation, perturbed cell fate, and a reduction in orga- of the brain. More recently, stem cells and iPSCs have been
noid volume reminiscent of microcephaly. differentiated into 3D organoid systems to study the develop-
ment of the intestine, retina, liver, kidney, and even the brain
(Koehler and Hashino, 2014; Lancaster and Knoblich, 2014).
INTRODUCTION These organoids are able to differentiate, self-organize, and
form distinct, complex, biologically relevant structures, thus
Zika virus (ZIKV) of the Flaviviridae family is an emerging mos- making them ideal in vitro models of development, disease
quito-borne virus originally identified in Uganda in 1947 (Driggers pathogenesis, and drug screening. Several groups have devel-
et al., 2016). Outbreaks of the virus have been previously recog- oped cerebral organoid models that generate functional cortical
nized in regions within Asia and Africa, including Malaysia, neurons and can recapitulate forebrain, midbrain, and hindbrain
Thailand, and Vietnam, and as far as Micronesia (Driggers et al., regions with functional electrophysiological properties to probe
2016; Hamel et al., 2015). ZIKV infects human skin and over the mechanisms of neurodevelopment, autism, and micro-
80% of ZIKV cases are asymptomatic or go unnoticed, while the cephaly (Camp et al., 2015; Eiraku et al., 2008; Lancaster
remaining cases typically exhibit mild fever, rash, and joint pain et al., 2013; Mariani et al., 2015; Nowakowski et al., 2016). Pre-
for a period of 7 days (Hamel et al., 2015; Petersen et al., 2016). vious studies have shown the utility of cerebral organoid models
However, with the increased incidence due to the current (from patient-derived iPSCs) in modeling microcephaly that

Cell Stem Cell 19, 18, July 7, 2016 2016 Elsevier Inc. 1
Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

results from a heterogeneous nonsense mutation in CDK5RAP2 in 1-month- and 2-month-old organoids when compared with un-
(Lancaster et al., 2013). The nonsense mutation altered the differentiated H9 cells (Figures 2A and S2A). Gene ontology
spindle orientation of radial glial cells, causing a severe analysis of the 1,642 differentially upregulated genes between or-
decrease in overall organoid size and premature differentiation ganoids and ESCs show significant enrichment of genes related to
of NPCs in the neuroepithelium. Because of the lack of outer neuron differentiation, development, cell morphogenesis, cell pro-
subventricular zone (OSVZ) in mice and unknown relevance of jection, axonogenesis, pattern specification, and regionalization,
ZIKV in mice, here we used human ESC (hESC)-derived cere- confirming the previously shown immunostaining results (Fig-
bral organoids to investigate the role of ZIKV in microcephaly. ure 2B). On the other hand, RNA-seq analysis shows enrichment
Here we show that cerebral organoids generated from hESCs of cell-cycle- and mitosis-related pathways in the 1,584 differen-
mimic the developing fetal brain and develop malformations tially expressed downregulated genes during organoid formation,
and severely inhibited growth following ZIKV inoculation. By as expected. Pathway analysis of differentially expressed genes
analyzing the transcriptomic profile of developing organoids, showed functionally grouped networks of developmental, neuro-
we can draw parallels between the stunted development of genesis, transcriptional, metabolic, cell-cycle, and cytoskeletal
ZIKV-infected organoids and TLR3-mediated dysregulation of genes in cerebral organoid development (Figure 2C). In addition,
neurogenesis and axon guidance. transcriptome analyses of organoids 1 month and 2 months in cul-
ture reveal an increase in genes related to visual perception, sen-
RESULTS sory and stimulus perception, phototransduction, and cognition,
indicating the early formation of immature retinal tissue as cerebral
Cerebral Organoids Display Regionalization and organoids further develop (Figures 2D and 2E).
Cortical Differentiation RNA-seq transcriptome data was then analyzed to contextu-
To model ZIKV infection in vitro in physiologically relevant alize cerebral organoids in terms of fetal brain development. Us-
models, cerebral organoids were generated from H9 hESCs us- ing the BrainSpan database of human brain transcriptomes, we
ing published protocols (Lancaster et al., 2013) with slight mod- calculated the Spearmans correlation between in vitro differen-
ifications described in the Experimental Procedures section. To tiated cerebral organoids with post-mortem human fetal brain
generate cerebral organoids, we formed embryoid bodies from tissues to further assess their age and regionalization (Figures
ESCs using the hanging drop method, and they differentiated 2F and 2G and S2B) (Kang et al., 2011). Based on these ana-
to form neuroectodermal tissue in three dimensions (Figure 1A). lyses, organoids showed significant correlation great than 0.5
Cerebral organoids display complex, self-organized internal between post-mortem neocortex (temporal, parietal, and occip-
morphology with fluid-filled ventricle-like structures similar to ital), ganglionic eminence (medial, lateral, and caudal), cere-
the developing cerebral cortex (Figures 1A and 1B). Immunohis- bellum, primary motor sensory cortex, upper rhombic lip, and
tochemistry for TUJ1 and SOX1 identifies regional specificity dorsal thalamus. In addition, organoids were correlated with
of neuronal and NPC populations, respectively (Figures 1C post-mortem fetal tissues ranging in age from 8 weeks post-
and S1). NESTIN-positive cells exhibit elongated morphology conception to 21 weeks post-conception. These data indicate
around cavities. TUJ1 staining shows broad neuronal expression that the organoid tissues most resemble early trimester fetal
throughout the organoid tissue, while SOX1 NPCs are localized brain tissues 8-9 weeks post-conception (Figures 2G and
in internal cavities, thereby emulating the intricate, radially out- S2C). In all, immunohistochemistry and transcriptome analyses
ward migratory pattern of differentiating neurons from the inner suggest that hESC-derived cerebral organoids robustly and
multiplication zone of the fetal brain. reproducibly model early trimester fetal brains.
To determine the regionalization and extent of cortical differ-
entiation and expansion, we immunostained organoids for ZIKV Infection Abrogates Organoid Growth
markers of early forebrain, hippocampus, dorsal cortex, and in- To determine the effect of ZIKV on fetal brain tissue in vitro,
terneurons (Figure 1C). PAX6 and FOXG1 expression highlight mouse neurospheres and early day 10 human cerebral organoids
the discrete organization of early forebrain tissue formation were infected with the prototype MR766 ZIKV strain originating
and specification to the ventral telencephalon. In addition, from Uganda, isolated from monkeys, and expanded in Vero
EMX1 expression shows subregionalization of dorsal cortex re- cells (Figure 3A). Mouse neurospheres were utilized because of
gions around cavities and throughout the intermediate zone. the large sample size and previous data suggesting robust reser-
PROX1 indicates differentiation of a portion of cerebral organoid voirs of ZIKV viral infection and ZIKV production in mouse brain
tissue to the hippocampus while CALB2 signifies the maturation tissues and their ability to recapitulate neurodegenerative phe-
of organoid NPCs into hippocampal calretinin-expressing inter- notypes in vivo (Lazear et al., 2016; Rossi et al., 2016). ZIKV-in-
neurons. To confirm functional neural activity in cerebral organo- fected neurospheres exhibited significantly attenuated growth
ids, fluctuations in cytosolic calcium content were analyzed by relative to control mock-treated samples (Figures 3B and 3C).
calcium dye imaging in response to glutamate (Figure 1D). To further confirm the negative effect of ZIKV on neurodevel-
opment, immature day 10 human organoids were utilized for
Cerebral Organoids Recapitulate Early Fetal Brain ZIKV infection because it coincides with the emergence of the
Development neuroepithelial layer and transition from embryoid body to cere-
Next, we compared the coding and non-coding transcriptome of bral organoid. Cerebral organoid growth was tracked over 5 days
H9 hESCs and their derived cerebral organoids to further charac- post-infection to monitor organoid growth and development. At
terize the cerebral organoid models. 3,226 and 3,357 significantly day 5 post-infection, healthy mock-treated cerebral organoids
differentially expressed genes with fold change > 2 were identified showed an average of 22.6% increase in growth while

2 Cell Stem Cell 19, 18, July 7, 2016


Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

Figure 1. Characterization of Cerebral Organoids Reveals Recapitulation of Fetal Brain Regions


(A) Bright-field image of a representative organoid shows development of the neuroepithelial layer. Scale bar represents 250 mm.
(B) DAPI-stained organoid shows complex inner morphology including ventricle-like structures from 30-day-old organoids. Scale bar represents 250 mm.
(C) Organoids immunostained for neuronal cells (TUJ1+) and NPCs (SOX1+). TUJ1 shows generalized neuronal differentiation while NPCs are localized near inner
ventricle-like structures in 30-day-old organoids. Immunostaining for forebrain (PAX6), dorsal cortex (EMX1), hippocampus (PROX1), and interneurons (CALB2)
show differentiation of organoids into discrete brain regions. Also see Figure S1. Scale bar represents 100 mm.
(D) Calcium dye imaging of cerebral organoids using Fluo-4 shows functional neural activity.

ZIKV-infected organoids significantly decreased by 16%, thus ZIKV-infected NESTIN-positive cells suggests an unhealthy
resulting in a net 45.9% difference in size on average (Figures state and activation of apoptotic processes. Since NPCs and
3D and 3F). The viral kinetics indicate a significant increase in radial glial cells may be susceptible to ZIKV and infection,
viral copy number 2 days post-infection, which is reflected in RT-qPCR of organoid supernatant reveals ZIKV replication and
the rate of change in organoid size after day 2 post-infection (Fig- permissiveness in NPCs and radial glial cells (Figure 3E). Taken
ures 3E and 3F). together, these results demonstrate that ZIKV abrogates neuro-
To probe the effect of ZIKV in fetal brain development and in development by targeting the NPC population.
NPCs, we cryosectioned and immunostained cerebral organoids
for markers of NPCs (NESTIN) and ZIKV envelope (ZIKVE) ZIKV Attenuates Organoid Growth by TLR3 Activation
expression. We observed strong co-localization of ZIKVE in and Regulation of Apoptosis and Neurogenesis
NESTIN-positive cell populations compared to NESTIN-negative Pathways
cells, indicating that ZIKV infects NPCs in organoid models (Fig- Previous studies have shown that ZIKV and other flaviviruses
ure 3G) (Tang et al., 2016). The non-elongated cell morphology of activate TLR3 in human skin fibroblasts (Hamel et al., 2015;

Cell Stem Cell 19, 18, July 7, 2016 3


Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

Figure 2. RNA Map of Cerebral Organoid Development


(A) Heat map of transcriptome analysis from hESCs (Group 1) and cerebral organoids after 1 month (Group 2) and 2 months (Group 3) in culture shows 3,226 and
3,357 significantly differentially expressed genes with fold change > 2, p < 0.05. See also Figure S2A and Table S1.
(B) Gene ontology analysis shows top 10 more enriched terms for upregulated (top) and downregulated (bottom) genes during cerebral organoid differentiation.
(C) Grouped functional pathway analysis of differentially expressed genes during organoid formation.
(D) Heat map of differentially expressed genes between organoids 1 month (Group 2) and 2 months (Group 3) in culture. Group 1 represents hESCs.
(E) Gene ontology analysis of differentially expressed genes in organoids 1 and 2 months old suggest the formation of retinal tissue.
(F and G) Spearmans correlation heat map of cerebral organoid transcriptomes compared with regions of the fetal brain and age in post-conception weeks (pcw).
See Table S2 for heat map brain region legend. Also see Figures S2B and S2C.

Tsai et al., 2009). Interestingly, TLR3 has been implicated in mock-treated organoids (n > 100 neurospheres per group) (Fig-
many neuroinflammatory and neurodegenerative disorders, ures 4A and 4B and S3A). To further reinforce the idea that TLR3
including those in NPCs (Cameron et al., 2007; Lathia et al., plays a key role in ZIKV-mediated microcephaly, neurospheres
2008; Okun et al., 2010, 2011). TLR3 is upregulated in cerebral were inoculated with ZIKV with TLR3 competitive inhibitor (Fig-
organoids and neurospheres after ZIKV infection as shown by ures 4B and S3B). We observed a statistically significant differ-
RT-qPCR analysis (Figures 3E and S3C). To investigate the ence between ZIKV-treated neurospheres with and without
link between ZIKV-mediated TLR3 activation and dysregulation TLR3 inhibitor, but no statistical significance between mock
of neurogenesis and apoptosis, we investigated the effect of and ZIKV+inhibitor groups.
TLR3 agonist poly(I:C) and a thiophenecarboxamidopropionate To validate our hypothesized link between ZIKV-mediated
compound that acts as a direct, competitive, and high-affinity TLR3 activation and dysregulation of neurogenesis and apoptosis
inhibitor of TLR3 inhibitor on mouse neurospheres and human in an orthogonal human model, organoids were treated with TLR3
organoids. To determine the effect of TLR3 activation, neuro- competitive inhibitor in the presences of ZIKV. Although there still
spheres were challenged with poly(I:C) and exhibited a statisti- appeared to be cell death and disruption of the developing neuro-
cally significant decrease in overall neurosphere size relative to epithelium characterized by the non-smooth outer surface of the

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Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

Figure 3. ZIKV Results in Attenuated Organoid Growth in Cerebral Organoids and Neurospheres through Activation of TLR3
(A) ZIKV-infected Vero cells for virus expansion. Vero cells were seeded and infected at MOI of 1 and viral supernatant was collected 48 hr post-inoculation. Scale
bar represents 100 mm.
(B) Bright-field images of mouse neurospheres at Day 0 and Day 1 post-inoculation with ZIKV. Scale bar represents 250 mm.
(C) Immunohistochemistry shows that ZIKV can robustly infect mouse neurospheres. Scale bar represents 50 mm.
(D) Representative bright-field images of individual human cerebral organoids treated with ZIKV over time. Scale bar represents 250 mm.
(E) ZIKV viral copy count in organoid supernatant quantified by one-step RT-qPCR after ZIKV infection shows organoid susceptibility and viral permissiveness.
***p < 0.001, Students t test. Data represent mean SEM.
(F) Quantification of organoid size over time with and without ZIKV infection. Bars represent the min, average, and max relative organoid size. Individual organoids
were measured over time relative to their respective Day 0 size from n = 5 organoid samples. *p < 0.05, Students t test.
(G) Representative images of ZIKV-treated organoids stained for ZIKV envelope protein and Nestin. Scale bar represents 100 mm.

organoid, the TLR3 competitive inhibitor attenuated the severe esis and apoptosis in organoid development, NTN1 and EPHB2
ZIKV-mediated apoptosis and organoid shrinkage seen in ZIKV- expression levels were analyzed by RT-qPCR with ZIKV+/ in-
only-treated organoids (Figure 4C). These data strongly suggest hibitor or poly(I:C) stimulation (Figures 4E4G). Consistent with
that TLR3 may play a pivotal role in the ZIKV-associated ZIKV infection, poly(I:C) treatment of organoids reduced NTN1
phenotype. and EPHB2 expression (Figure 4F). In addition, TLR3 competitive
To determine the role of TLR3 activation in neurodegeneration, inhibitor reversed the downregulation of NTN1 and EPHB2 by
we compared differentially expressed genes involved in cerebral ZIKV infection (Figure 4G). All together, these data suggest that
organoid formation identified by RNA-seq with differentially ex- ZIKV perturbs a TLR3-regulated network controlling neurogene-
pressed genes following poly(I:C)-challenged TLR3 activation sis and apoptotic pathways (Figure 4H).
(data not shown) and found 41 genes in common (Figure 4D).
Pathway analysis was performed to identify potential pathways DISCUSSION
by which ZIKV may regulate neurogenesis. Intriguingly, from
these 41 genes, only networks relating to positive regulation of Here we report the generation and application of hESC-derived
nervous system development and regulation of synapse struc- cerebral organoids for modeling and analyzing the relationship
ture or activity were significantly enriched (Figure 4D). To validate between ZIKV and microcephaly. To properly model the com-
the proposed genes regulated by TLR3 that modulate neurogen- plexities of the fetal brain, we employed 3D organoid models

Cell Stem Cell 19, 18, July 7, 2016 5


Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

Figure 4. ZIKV Induces TLR3 and Regulates Pathways Involved in Apoptosis and Neurogenesis
(A) Bright-field images of mouse neurospheres at Day 0 and Day 1 post-inoculation with ZIKV with or without TLR3 competitive inhibitor, or TLR3 agonist poly(I:C).
Scale bar represents 250 mm.
(B) Neurospheres show significant change in size 1 day post-inoculation with ZIKV with or without TLR3 competitive inhibitor, or TLR3 agonist poly(I:C), as
quantified by ImageJ. Box and whisker plots show 1090 percentile. *p < 0.05, ***p < 0.001, n.s. = not significant, Students t test.
(C) Representative bright-field images of individual human cerebral organoids treated with ZIKV with or without TLR3 competitive inhibitor. Scale bar represents
250 mm.
(D) Schematic of target selection for RT-qPCR analyses. We analyzed differentially expressed genes involved in organoid formation (from Figure 2A) and TLR-
regulated genes (data not shown) to identify common pathways activated upon ZIKV infection. The two significantly enriched pathways from this dataset were
positive regulation of nervous system development and regulation of synapse structure or activity.
(E) RT-qPCR analysis of TLR3 upregulation in organoids mock and ZIKV treated. Error bars represent SEM.
(F) RT-qPCR analysis of differentially expressed genes, NTN1 and EPHB2, involved in TLR3 activation and neurogenesis in organoids treated with TLR3 agonist
poly(I:C). Error bars represent SEM. *p < 0.05. **p < 0.01, Students t test.
(G) RT-qPCR analysis of NTN1 and EPHB2 expression in human organoids upon ZIKV and ZIKV+ TLR3 competitive inhibitor. *p < 0.05, ***p < 0.001, Students t
test. Data represent mean SEM.
(H) Model for ZIKV infection and TLR3-mediated downregulation of regulators of neurogenesis and upregulation of pro-apoptotic pathways in NPCs.

capable of recapitulating regions of the developing neocortex, Neurological manifestations, such as viral encephalitis, have
ganglionic eminence, and retinal tissue as evidenced by immuno- previously been linked to other viruses of the Flaviviruses genus
histochemistry and transcriptomic analyses. These in vitro cere- (Sips et al., 2012). TLR3 has been linked to neurodegenerative
bral organoid models present a scalable and reproducible model disorders and negative regulation of axonogenesis, as well as
for neurodevelopmental and neurodegenerative studies. We then dengue and ZIKV infection, so we hypothesized that ZIKV
treated organoids with prototype MR766 ZIKV to understand the activates the TLR3 pathway in NPCs, thereby leading to pro-
phenotypic and transcriptomic response during early stage neu- apoptotic pathway activation and/or dysregulation of cell fate de-
ral development. Organoids treated with ZIKV showed significant cisions (Cameron et al., 2007; Hamel et al., 2015; Okun et al., 2010,
decrease in the neuroepithelium and overall organoid size. 2011; Tsai et al., 2009; Yaddanapudi et al., 2011). As seen in

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Please cite this article in press as: Dang et al., Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate
Immune Receptor TLR3, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.04.014

microcephaly, dysregulated cell fate, self-renewal, and apoptotic University of California, San Diego. All animal work was approved by the Insti-
pathways in NPCs may contribute to the microcephaly pheno- tutional Review Board at the University of California, San Diego and was per-
formed in accordance with Institutional Animal Care and Use Committee
type. TLR3 is highly expressed in early brain development and
guidelines. H9 hESCs were detached from their feeder layer using 1 mg/ml
decreases as the NPC population differentiates and the brain ma- collagenase for 1520 min and 0.5 mg/ml dispase for an additional 15 min.
tures (Lathia et al., 2008). This temporally sensitive expression of Wells were washed with media to collect floating undifferentiated hESCs
TLR3 during early brain development may contribute to the and colonies were dissociated using Accumax at 37 C for 10 min to generate
trimester-specific response of fetal brains to ZIKV infection. In- a single-cell suspension. At Day 0, embryoid bodies were formed using the
duction of TLR3 has been shown to trigger apoptosis by inhibiting hanging drop method with 4,500 cells/drop in DMEM/F12 media supple-
mented with 20% knockout serum replacement, 4 ng/ml bFGF, NEAA, and
Sonic Hedgehog and Ras-ERK signaling in NPCs and plays a role
glutamine. After 2 days of hanging drop culture, embryoid bodies were trans-
in retinopathy (Shiose et al., 2011; Yaddanapudi et al., 2011).
ferred to sterile petri dishes with refreshed media. After 6 days in culture,
Moreover, TLR3 has been connected to the elevated risk of neuro- embryoid bodies were transferred to new petri dishes containing neural induc-
pathological dysfunction resulting from maternal infection using tion media consisting of DMEM/F12, 1:100 N2 supplement, NEAA, glutamine,
TLR3-deficient mouse models (De Miranda et al., 2010). Based and 1 mg/ml heparin until day 11. At day 11, organoids were transferred to
on these data, TLR3 likely plays a dual role that is cell type specific Matrigel droplets and cultured in 1:1 mixture of DMEM/F12 and Neurobasal
in which potent downstream anti-viral responses are activated in medium supplemented with 1:100 B27 without vitamin A, 1:200 N2, NEAA, in-
sulin, beta-mercaptoethanol, and glutamine. Organoids were then transferred
addition to tangential dysregulation of signaling networks direct-
to stir flask bioreactors for long-term growth on day 15 in the same differenti-
ing apoptosis and neurogenesis. ation media except with the addition of retinoic acid and vitamin A. Media was
By comparing changes in the transcriptomic profiles during ce- changed every 3 days.
rebral organoid formation and after TLR3 activation by poly(I:C),
we identified several candidate genes (NTN1, EPHA3, ADGRB3, ZIKV Expansion and Infection
EPHB2, SLITRK5, SYT11, and GRIK2) that may be responsible To expand the prototype MR766 virus, Vero cells were inoculated with virus at
for depletion of the NPC population and the subsequent micro- MOI of 1 in E-MEM 10% FBS medium. Media was changed 24 hr after inocu-
lation and viral supernatant was collected at 48 hr post-inoculation. Viral titer
cephaly phenotype through pathway analysis. Many of these
was assessed using iScript One Step RT-PCR kit (Bio-Rad) and viral copy
TLR3-activated genes have been implicated in early brain cell number was calculated based on a standard curve of in vitro transcribe viral
fate decisions. Netrin1 is a secreted protein that works in conjunc- transcripts. Organoids were inoculated with ZIKV at MOI of 1.
tion with its dependence receptor DCC (Deleted in Colorectal
Carcinoma) to regulate various pathways involved in axon guid- ACCESSION NUMBERS
ance, apoptosis, neural cell death, and cellular reprogramming
(Bin et al., 2015; Furne et al., 2008; Ozmadenci et al., 2015). The The GEO accession number for the RNA-seq data reported in this paper is
role of NTN1 has been shown in vivo in floxed and null NTN1 GEO: GSE80264.
mice. Complete loss of the gene results in severe axon guidance
defects and death shortly after birth (Bin et al., 2015). Evidence SUPPLEMENTAL INFORMATION
suggests that NTN1 interacts with DCC to limit apoptosis but
Supplemental Information for this article includes three figures, two tables, and
additional data has shown that the absence of NTN1 can also
Supplemental Experimental Procedures and can be found with this article on-
upregulate DCC, thus additionally triggering a pro-apoptotic line at http://dx.doi.org/10.1016/j.stem.2016.04.014.
cascade (Bin et al., 2015). In addition to NTN1, the membrane-
bound receptor tyrosine kinase EPHB2 has been shown to be AUTHOR CONTRIBUTIONS
integral to fetal brain development by regulating angiogenesis,
vasculogenesis, and neurogenesis. EPHB2 modulates radial J.D. designed and performed experiments, analyzed the data, and wrote the
migration, proliferation, and cell fate of NPC in the subventricular manuscript; S.K.T. performed experiments, analyzed the data, and partici-
pated in figure preparation; G.L. performed experiments and analyzed the
zone (Chumley et al., 2007; Katakowski et al., 2005). Interestingly,
data; Y.Q. analyzed the data; V.S.P. performed experiments and data analysis;
NTN1 and EPHB2 have been shown to work synergistically A.M.E. contributed to data analysis; and T.M.R. contributed to the concept
through the Src family kinase-signaling pathway during neural and design, data analysis and interpretation, and manuscript writing. All au-
circuit assembly (Poliak et al., 2015). However, further mecha- thors approved the final version of this manuscript.
nistic studies will be required to validate the significance and
underlying molecular mechanisms by which these putative genes ACKNOWLEDGMENTS
potentially cause viral-mediated microcephaly. Nonetheless,
We thank Steve Head and the staff of the Next Generation Sequencing core
our results present evidence that TLR3 activation of multiple ge-
facility at The Scripps Research Institute for help with the HT-seq and data
netic hubs regulating axonogenesis, cell proliferation, and anti-
analysis, and we thank members of the Rana laboratory for helpful discussions
apoptotic pathways within NPCs may strongly contribute to the and advice. This work was supported in part by grants from the NIH.
ZIKV-mediated microcephaly phenotype using robustly repro-
ducible and scalable human cerebral organoid models. Received: April 6, 2016
Revised: April 18, 2016
Accepted: April 27, 2016
EXPERIMENTAL PROCEDURES Published: May 6, 2016

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