. 2017 Oct 10;21(2):517-532.

doi: 10.1016/j.celrep.2017.09.047.

Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection

Momoko Watanabe¹, Jessie E Buth¹, Neda Vishlaghi¹, Luis de la Torre-Ubieta², Jiannis Taxidis³, Baljit S Khakh⁴, Giovanni Coppola², Caroline A Pearson¹, Ken Yamauchi¹, Danyang Gong⁵, Xinghong Dai⁵, Robert Damoiseaux⁵, Roghiyh Aliyari⁶, Simone Liebscher⁷, Katja Schenke-Layland⁸, Christine Caneda⁹, Eric J Huang¹⁰, Ye Zhang⁹, Genhong Cheng⁶, Daniel H Geschwind¹¹, Peyman Golshani², Ren Sun⁵, Bennett G Novitch¹²

Affiliations

¹ Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Center for Autism Research and Treatment and Program in Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Physiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
⁸ Department of Cardiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, 72076 Tübingen, Germany; Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, 70569 Stuttgart, Germany.
⁹ Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA.
¹¹ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Center for Autism Research and Treatment and Program in Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: bnovitch@ucla.edu.

PMID: 29020636
PMCID: PMC5637483
DOI: 10.1016/j.celrep.2017.09.047

Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection

Momoko Watanabe et al. Cell Rep. 2017.

. 2017 Oct 10;21(2):517-532.

doi: 10.1016/j.celrep.2017.09.047.

Authors

Affiliations

¹ Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Center for Autism Research and Treatment and Program in Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Physiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
⁸ Department of Cardiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, 72076 Tübingen, Germany; Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, 70569 Stuttgart, Germany.
⁹ Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA.
¹¹ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Center for Autism Research and Treatment and Program in Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Neurology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: bnovitch@ucla.edu.

PMID: 29020636
PMCID: PMC5637483
DOI: 10.1016/j.celrep.2017.09.047

Abstract

The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions.

Keywords: Zika virus; cerebral cortex; differentiation; embryonic stem cell; human brain; neural development; neural stem cell; neurogenesis; organoid.

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Figures

**Figure 1. Establishment of highly reproducible cortical organoid methods that recapitulate human development in vivo**
(A) Cerebral organoids immunostained for telencephalic (FOXG1/LHX2/PAX6), apical membrane (NCAD/APKC), progenitor (NESTIN), and cell death (clCASP3) markers. (B) Percentage of FOXG1⁺ and LHX2⁺ cells out of total live cells per organoid. hESC lines, H9 and UCLA1 (U1), and hIPSC lines, HiPS2 (Hi2) and XFiPS (XF). H9 n = 9, n = 3 for other lines. Data are represented as mean ± SEM. (C) The perimeters of W2.5 organoids derived from H9 across nine experiments plotted as mean ± SEM. All (blue circle) indicates the summation of all samples plotted as mean and 95% confidence intervals. No significance between samples by one-way ANOVA. (**D-E**) Comparison of FBS versus B27 media components, showing CTIP2⁺ neurons, TBR2⁺ intermediate progenitor, PAX6⁺ progenitor, and pVIM⁺ dividing RG cell markers n=3. Data are represented as mean ± SEM. (F) Laminar growth of cerebral organoids showing SOX2⁺ progenitor, TBR2⁺ intermediate progenitor, and TBR1⁺ neurons. (G) Average number of cells per field ± SEM at various time points. n=3. (H-I) Comparative immunohistochemical analyses of W8 cerebral organoids (H) and GW14 human fetal cortex (I). Scale bars: A FOXG1 and W5 FOXG1/LHX2/NCAD 500 μm; A W2.5 FOXG1/LHX2/NCAD, D, H, I, 100 μm; D HOESCHT, 1mm; F, 50 μm. See also Figures S1, S2, and S3.

**Figure 2. Transcriptomic analyses confirm that cerebral organoids faithfully recapitulate fetal brain development**
(A) Hierarchical clustering of cerebral organoid gene expression from four time-points (W5, W8, W11, W14), each with three replicates. Heatmap colored according to inter-sample Pearson coefficient from highest (dark red) to lowest (blue) correlation. (B) Predicted regional identity of cerebral organoids by CoNTExT analysis. (C) Predicted fetal developmental period (defined in table) for W5 and W14 cerebral organoids. (**D-E**) TMAP analysis of developmental (D) and laminar (E) transitions, comparing the progression of W5-W14 organoid to that of indicated pairs of developmental periods. Schematic of laminar organization shown in (E). VZ=ventricular zone, SVZ=subventricular zone, IZ=intermediate zone, SP=subplate, CPi=inner cortical plate, CPo=outer cortical plate, MZ=marginal zone, and SG=supragranular layer. Genes upregulated (lower left corner) or downregulated (upper right corner) from in vitro W5 to W14 compared to in vivo transitions. (F) WGCNA analysis of developmental processes conserved in organoids versus fetal tissue. Well-preserved modules have preservation Z-score ≥ 4 (dotted red line). See also Table S1.

**Figure 3. LIF/STAT3 activation increases the formation of bRG cells and stimulates astrogliogenesis**
(**A-C**) Cerebral organoids immunostained for general RG (PAX6/SOX2), bRG (HOPX), IP (TBR2), and dividing RG (pVIM) cell markers. (D) Thickness of the SVZ in μm and number of bRG. bRG were counted as abventricular PAX6⁺ and pVIM⁺ cells. Data are represented as mean ± SEM. (E) Antibody staining for GFAP+/HEPACAM⁺ astrocytes or CTIP2⁺ excitatory neurons in control versus LIF-treated organoids. (F) Cerebral organoids and human fetal cortex immunostained for CTIP2⁺ lower layer neurons, BRN2⁺ upper layer neurons, and LAMININ⁺ basement membrane production. Arrowheads denote blood vessels. (G) W22 organoids with or without LIF treatment immunostained for the cell death marker clCAS3, CTIP2⁺ or TBR1⁺ lower layer neuronal markers, and SATB2⁺ or CUX1⁺ upper layer neuronal markers. (H) Relative cortical plate position of CTIP2+, TBR1+, SATB2+, or CUX1⁺ neurons in W22 organoids with or without LIF. Values represent median ± 95%CI. Total number of neurons counted from 3 independent experiments, Control: CTIP2⁺ n=,837 TBR1⁺ n=1088, SATB2⁺ n=836, CUX1⁺ n=718; LIF: CTIP2⁺ n=931, TBR1⁺ n=1038, SATB2⁺ n=765, CUX1⁺ n=546; n.s. no significance, ****p < 0.0001, Statistical analysis compares indicated neuronal markers to the CTIP2⁺ group, Kruskal-Wallis test with Dunn's Correction. All scale bars: 50 μm. See also Figures S4 and S5.

**Figure 4. Action potentials and spontaneous network activities in cerebral organoids**
W11-W12 organoid slice cultures were prepared for electrophysiological recordings, calcium imaging, and immunostaining. (A) Representative TTX-sensitive spike trains upon current stimulation. n=12/24 cells had APs from 3 independent experiments, (B) rectified TTX-blocked sodium currents, and (C) potassium currents. Data are represented as mean ± SEM. (D) Physiological properties of cells with and without APs (membrane potential, membrane resistance, capacitance, peak sodium currents, and peak potassium currents) n=24. Data are represented as mean ± SEM. (**E-F**) Cerebral organoids immunostained for CTIP2⁺ excitatory neurons, CaMK2α⁺ mature neuronal markers, and calcium indicator AAV1-SYN∷GCaMP6f (anti-GFP staining). (**G-G′**) 3D reconstruction of axosomatic and axodendritic contacts made between SYN∷ChR2-YFP labeled neurons immunostained for the excitatory synaptic marker VGLUT1. (H) Normalized GCaMP6f calcium traces from several neurons of an organoid imaged with two-photon microscopy. Black dots indicate action potential time-points extracted from the fast calcium transients (I) Matrix of spiking correlation values between all cell pairs in an organoid. Lighter colors indicate higher correlations. Autocorrelation and non-significantly correlated pairs were set to zero. (J) Examples of calcium traces from significantly correlated cell pairs. See also Figures S6 and Movie S1. See also Table S4 for statistics. Scale bars: E, F, 100 μm; G, G′, 10 μm.

**Figure 5. Infection of cerebral organoids with ZIKV leads to widespread progenitor apoptosis and overall growth restriction**
(**A-B**) Cerebral organoids immunostained for the ZIKV E protein, candidate ZIKV receptor (AXL), and PAX6+/SOX2⁺ progenitors, TBR2⁺ IP, CTIP2⁺ excitatory neurons, or AQP1⁺ choroid plexus markers. (C) VZ/SVZ regions of W8 cerebral organoids immunostained for the additional ZIKV candidate receptors TYRO3, MER, TIM1, and CD209. (D) Top, percentage of all ZIKV⁺ cells also labeled with indicated markers. Bottom, percentage of each cell type also infected with ZIKV. n=3 independent experiments. (E) Cerebral organoids stained with propidium iodide (PI) to label dead cells and the general nuclear stain. (F) Cell death was measured as PI intensity relative to HOECHST, area in mm2, and perimeter in mm. Mock n=23, ZIKV n=26. (G) Cerebral organoids collected 14 dpi immunostained for ZIKV, SOX2, TBR2, CTIP2, and clCASP3. (H) ZIKV infection was scored as the percentage of each cell type displaying ZIKV envelope staining. Cell death was scored as the percentage of each cell type exhibiting clCASP3 staining. n=3. Data in all charts are represented as mean ± SEM. Scale bars: A, 50 μm; B, 500 μm; E top, 200 μm; E brightfield, 1 mm; G, 100 μm. See also Figure S7.

**Figure 6. Immune response and programmed cell death in cerebral organoids infected with ZIKV**
(A) Immunostaining of W8 organoids infected with ZIKV for the indicated number of days. Scale bar: 100 μm. (B) Percentage of cells per field positive for clCASP3 out of total cells. n=3. Data are represented as mean ± SEM. (C) Gene ontology (GO) annotation of the upregulated and downregulated transcripts in W8 organoids after 3 and 5 dpi with ZIKV compared to mock infected organoids by RNA sequencing. See also the lists of the top 50 upregulated (Table S2) and downregulated genes (Table S3). Cell color represents the –log10 (p-value) for overrepresentation of each GO category within up or downregulated genes. See also Figure S8.

**Figure 7. Identification of compounds capable of mitigating the teratogenic effects of ZIKV infection**
Cerebral organoids immunostained for ZIKV and cell death marker clCASP3 at 7 dpi in the absence or presence of (A) 25HC, (B) R428, (C) Duramycin, and (D) Ivermectin. Scale bar: 100 μm. RT-qPCR analysis of ZIKV expression. Plots represent expression levels normalized to ZIKV-vehicle average. 25HC n=6, R428 n=5, Duramycin n=4, and Ivermectin n=4. Percentage ZIKV⁺ or clCASP3⁺ cells out of total cells per field. ZIKV: 25HC: n=6, R428: n=3-4, Duramycin: n=7, and Ivermectin: n=8; clCASP3: 25HC: n=6, R428: n=3-4, Duramycin: n=3-8, and Ivermectin: n=8-9. Data are represented as mean ± SEM. See also Figure S9 and Table S5 for statistical analysis details.

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Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection

Affiliations

Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection

Authors

Affiliations

Abstract

Figures

References

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Medical

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