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, 165 (5), 1238-1254

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure

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Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure

Xuyu Qian et al. Cell.

Abstract

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

Figures

Figure 1
Figure 1. SpinΩ Bioreactor-based Forebrain Organoid Culture System
(A) Computer-Aided-Design drawings of 12-well version SpinΩ bioreactor and individual parts. (B) Schematic diagram of forebrain organoid protocol and sample phase images at different stages. Scale bars: 200 μm. (C–D) Immunostaining of forebrain organoids at days 14, 63 and 84. Scale bars: 100 μm. Also see Figure S1 and Table S1.
Figure 2
Figure 2. Homogeneity of Early Stage Forebrain Organoids and Effects of BPA
(A) Sample images and quantification among multiple iPSC cell lines and clones for immunostaining of organoids at day 14. Scale bars: 100 μm. Values represent mean ± SEM (42–128 and 11- 30 total neural tube structures from at least 10 organoids each for the forebrain protocol and “intrinsic protocol”, respectively; ***P < 0.005, Student’s t-test). (B) Schematic drawing for SOX2+ ventricular zone (VZ) and TUJ1+ neuronal layer measurement in cortical structures (top panel) and box plot for relative VZ thickness in day 14 organoids. For each cortical structure, three measurements were taken at 45 degree angles to obtain the mean value. Relative VZ thickness is the ratio of VZ thickness to total thickness from ventricular surface to pial surface. The red dot indicates mean; upper and lower error bars in each box plot represent the top whisker (maximum value) and bottom whisker (minimum value), respectively (n = 30 and 15 cortical structures from at least 10 organoids each for the forebrain and “intrinsic protocol”, respectively). (C–D) Sample images of immunostaining of organoids at day 28 (C; Scale bars: 100 μm) and box plot for relative VZ thickness (D). Similar to (B) (n = 20 cortical structures from at least 10 organoids each). (E–F) Effect of BPA treatment of forebrain organoids from day 14 to 28. Shown are sample images of immunostaining of control and BPA-treated forebrain organoids (E) and quantification of relative VZ thickness (F) at day 28. Scale bar: 100 μm. Values represent mean ± SEM. (n = 21 cortical structures from at least 10 organoids; ***P < 0.0005, Student’s t-test). Also see Figure S2.
Figure 3
Figure 3. Organization and Marker Expression of Different Progenitor Zones and Cortical Neuron Subtypes
(A–C) Schematic representations and sample immunostaining images of forebrain organoids at days 28 (A), 56 (B) and 84 (C). Scale bars: 50 μm. Dash lines highlight a gap between oSVZ and iSVZ (C). PP: preplate; VZ: ventricular zone; MZ: marginal zone; CP: cortical plate; SVZ: subventricular zone; oSVZ: outer subventricular zone; iSVZ: inner subventricular zone. (D–G) Sample images of immunostaining of oRGC markers HOPX (D), FAM107A (E) and PTPRZ1 (G), and Ki67 (F) in day 84 forebrain organoids. Scale bars: 50 μm. (H) Schematic representation of marker expression for cortical neurons in the mature mammalian neocortex. (I) Sample images of immunostaining for preplate Cajal-Retzius cell marker REELIN and deep layer neuron markers CTIP2 and TBR1 in day 28 forebrain organoids. Scale bar: 50 μm. (J–K) Sample images of immunostaining for CTIP2, TBR1 and superficial layer neuron marker (SATB2) in forebrain organoids at days 56 (J) and 70 (K). Images shown in (K) are from consecutive sections for the same cortical structure. Scale bars: 50 μm. (L) Sample images of immunostaining for CTIP2, TBR1 and superficial layer neuron markers (SATB2, BRN2 and CUX1) in forebrain organoids at day 84. Images shown are from consecutive sections for the same cortical structure. Scale bar: 50 μm. (M) Sample image showing layer specification in forebrain organoids and quantification of the relative thickness of VZ, SVZ and CP at days 56 and 84 for two iPSC lines. For each cortical structure, three measurements were taken at 45 degree angles to obtain the mean. Values represent mean ± SEM (n ≥ 6 cortical structures from 6 organoids). Also see Figure S3.
Figure 4
Figure 4. Correlation of Global Transcriptomes between Forebrain Organoids and Fetal Human Brain Development
(A) Heatmap of Pearson’s correlation analysis of RNA-seq datasets from day 26, 40, 54 and 100 organoids and published datasets from 21 different human fetal organs (Roost et al., 2015). Shown are averaged values for biological replicates. (B) Heatmaps of Pearson’s correlation analysis of RNA-seq datasets among forebrain organoids at different stages and published transcriptome datasets of 3 different cortical subregions at 11 developmental stages from Allen Brain Atlas. See Figure S5C for comparison of all 16 different human brain regions. (C) Heatmap of gene expression dynamics from up-regulated genes between day 26 and later stages of organoid development. (D) Gene Ontology analysis of upregulated genes. Nine top terms (in terms of P-values) are shown. (E) Overlap of differentially expressed genes during organoid development with known schizophrenia related risk genes (from http://bioinfo.mc.vanderbilt.edu/SZGR/) and autism related risk genes (from https://gene.sfari.org/autdb/HG_Home.do). Overlapping genes are statistically significant (P < 0.001, chi-square test). Also see Figure S4 and Table S2
Figure 5
Figure 5. Functional Characterization of Forebrain Organoids
(A–D) Electrophysiological and morphological analyses of cells in forebrain organoids. Shown in (A) are sample current-clamp traces of a neuron firing a train of action potentials in response to 10 pA current injection. A hyperpolarizing step of - 5 pA is also shown. Shown in (B) are sample voltage-clamp traces showing currents in response to a ramp protocol (− 90 mV to 110 mV). Shown in (C) is a sample image of a neuron in a day 85 forebrain organoid labeled by GFP upon electroporation. The insert shows surface rendering of a dendritic spine structure on a GFP+ neuron with the pre-synaptic terminal labeled by SV2 staining in red. Scale bars: 50 μm. Shown in (D, left) are sample recording traces of sEPSCs and pharmacological blockade by DNQX. Identified sEPSC events are overlaid and the average sEPSC trace is shown. Also shown in (D, right) is the summary of the percentage of cells that exhibited detectable sEPSC events in organoids of different ages. (E) Calcium imaging analysis of cellular response to GABA application (10 μM). Day 100 organoids were loaded with Fluo-4. Shown in left panels are sample heat maps of GABA-induced fluorescence changes (ΔF/F) within the same region in the absence or presence of Bicuculline (Bicu. 50 μM). The color scale at the right indicates a ΔF/F range of 0 to 250%. Scale bar: 50 μm. Shown in the middle panel are calcium response curves for individual cells indicated in the heatmap. Shown in the right panel is the summary of ΔF/F in response to GABA in absence or presence of bicuculline. Values represent mean ± SEM (n = 43 neurons from 3 organoids). (F) Developmental shift of the percentage of cells in forebrain organoids that exhibit calcium rise in response to GABA (10 μM) and glutamate (20 μM). Value represent mean (n = 26, 77 and 69 neurons from 3 organoids at days 50, 80 and 100, respectively). (G–I) GABAergic neurons in forebrain organoids. Shown are sample images of immunostaining for GABA and VGLUT1 (G) and GABAergic neuron subtypes (I). Scale bars: 50 μm. Shown in (H) are sample recording traces of sIPSCs. Identified sIPSC events are overlaid and the average sIPSC trace is shown. (J–L) Sample images of immunostaining for astrocyte markers S100β (J) and GFAP (K) in organoids over 100 days. Scale bars: 50 μm. Also see Figure S5.
Figure 6
Figure 6. Generation of Midbrain and Hypothalamic Organoids
(A–E) Midbrain organoids from human iPSCs. Shown in (A) is a schematic diagram of the midbrain organoid protocol. Shown in (B) are sample images of day 18 organoids (Scale bars: 100 μm) and quantifications. Values represent mean ± SEM (n = 4 organoids each; *P < 0.05, Student’s t-test). Also shown are sample images of immunostaining of midbrain organoids at day 38 (C) and day 75 (D), and monolayer cultures 5 days after dissociation and plating of day 65 midbrain organoids (E). Scale bars: 50 μm. (F–H) Hypothalamic organoids. Shown in (F) is a schematic diagram of the hypothalamic organoid protocol. Shown are sample images of day 8 (G) and day 40 (H) organoids. Scale bars: 100 μm. Also shown in (H) is a summary of quantification for peptidergic neuronal markers expression in day 40 hypothalamic organoids from 2 iPSC lines. Values represent mean ± SEM (n = 3 organoids each). Also see Figure S6.
Figure 7
Figure 7. Modeling Impact of ZIKV Exposure using Forebrain Organoids
(A) Sample immunostaining images of forebrain organoids exposed to ZIKVM (1X) or mock-treated at day 14 for 24 hours and analyzed 10 days later (14+10). Scale bar: 100 μm. (B–C) Sample immunostaining images of forebrain organoids exposed to ZIKVM (1X) or mock-treated at day 14 for 24 hours and analyzed 18 days later (14+18) for CAS3 (B), or PH3 (C). Scale bars: 100 μm. Also shown are quantifications for the percentage of CAS3+ cells among the total number of nuclei stained by DAPI (B) and density of PH3+ cells within VZ (C). Values represent mean ± SEM (n = 5 organoids; *** P < 0.0005, Student’s t-test) (D) Sample immunostaining image of a forebrain organoid exposed to ZIKVM (1X) at day 28 for 24 hours and analyzed 4 days later (28+4) (Scale bars: 100 μm) and quantifications. Values represent mean ± SEM (n = 5 cortical structures from 3 organoids; *** P < 0.0005, Student’s t-test). (E–F) Forebrain organoids exposed to ZIKVM (1X or 0.25X) or mock-treated at day 28 for 24 hours and analyzed 14 days later (28+14). Shown are sample immunostaining images and quantification of cell proliferation and cell death in ZIKV-infected regions (E) and thickness of SOX2+ VZ layer and TUJ1+ neuronal layer (F). Scale bars: 100 μm. Values represent mean ± SEM (n = 5 cortical structures from 3 organoids; *P < 0.05; **P < 0.005, ***P < 0.0005, Student’s t-test). (G–J) Sample immunostaining images (top; Scale bars: 100 μm) and magnified views (bottom; Scale bars: 50 μm) of forebrain organoid exposed to ZIKVM (1X) at day 80 for 24 hours and analyzed 10 days (80+10; G–H) or 20 days later (80+20; I–J). Arrows in (J) point to ZIKV+HOPX+SOX2+ oRGC-like cells in the oSVZ region. Also shown in (I) are quantifications for the percentage of ZIKV+ cells among the total number of SOX2+ or CTIP2+ cells in the whole cortical structure. Values represent mean ± SEM (n = 7 cortical structures from 5 organoids, *P < 0.05, Student’s t-test). Also see Figure S7.

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