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. 2017 Jul;14(7):743-751.
doi: 10.1038/nmeth.4304. Epub 2017 May 10.

Fused Cerebral Organoids Model Interactions Between Brain Regions

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Free PMC article

Fused Cerebral Organoids Model Interactions Between Brain Regions

Joshua A Bagley et al. Nat Methods. .
Free PMC article

Abstract

Human brain development involves complex interactions between different regions, including long-distance neuronal migration or formation of major axonal tracts. Different brain regions can be cultured in vitro within 3D cerebral organoids, but the random arrangement of regional identities limits the reliable analysis of complex phenotypes. Here, we describe a coculture method combining brain regions of choice within one organoid tissue. By fusing organoids of dorsal and ventral forebrain identities, we generate a dorsal-ventral axis. Using fluorescent reporters, we demonstrate CXCR4-dependent GABAergic interneuron migration from ventral to dorsal forebrain and describe methodology for time-lapse imaging of human interneuron migration. Our results demonstrate that cerebral organoid fusion cultures can model complex interactions between different brain regions. Combined with reprogramming technology, fusions should offer researchers the possibility to analyze complex neurodevelopmental defects using cells from neurological disease patients and to test potential therapeutic compounds.

Figures

Figure 1
Figure 1. Ventral drug-treatment produces ventral-forebrain containing cerebral organoids.
(A) Schematic of cerebral organoid protocol with ventral drug-patterning application (2.5µM IWP2 and 100nM SAG) during the neural induction step. (B) Schematic of a human coronal brain slice indicating the regional expression of the patterning markers used for qPCR/IHC analysis. (C) qPCR analysis of the expression of different brain regional markers showing an increase in ventral and decrease in dorsal forebrain identity in ventral cerebral organoids. Values are plotted as relative expression level (2-ΔCt) to the reference gene TBP. Each data point corresponds to a pooled batch of 8-10 organoids. Data is represented as mean±SD, and statistical significance was tested using the student’s t-test (df=9) for dorsalUnt (n=6 batches) versus ventral-(IWP2+SAG) (n=5 batches) treatment. (D-E) Widefield images of immunostaining analysis of dorsalUnt and ventral cerebral organoids indicating the ventral-forebrain identity of ventral organoids (D), and the dorsal-forebrain identity of dorsalUnt organoids (E). Scale bars are 200µm.
Figure 2
Figure 2. Fusion of cerebral organoids allows cell migration between ventral and dorsal forebrain tissue.
(A) The experimental outline of the cerebral organoid fusion co-culture method. (B) Representative widefield images at different stages throughout the organoid fusion procedure. (C) Tile-scan image of an immunostained cryosection of a ventral::dorsalCycA organoid fusion indicates the combination of ventral (NKX2-1+) and dorsal (TBR1+) regions. (D) Tile-scan image of an immunostained cryosection of a ventral/GFP+::dorsalCycA/tdTomato+ organoid fusion indicates robust migration from ventral (GFP+) to dorsal tissue, but little migration from dorsal (tdTomato+) into ventral tissue. (E) Immunostained ventral::dorsalCycA organoid fusion cryosections from organoids of different ages shows the time course of GFP+ cells migrating from ventral into dorsal tissue. (E) The GFP+ cell density in dorsal tissue was quantified in cryosections from 32 (n=3 organoids) 46 (n=3), 58 (n=4), and 80 (n=4) day-old organoids. The data is presented as mean±SD and statistical significance tested using the one-way ANOVA [F(3,10)=12.59, p=0.0010] with posthoc Tukey’s test for between group comparisons. Scale bars are 500µm.
Figure 3
Figure 3. Mixing the tissue components of cerebral organoid fusions indicates the most robust migration from ventral into dorsal regions.
(A) Cerebral organoid fusions were created containing different combinations of ventral (V), dorsalUnt (Duntr) or dorsalCycA (DCycA) treated tissue. The components were labeled with either GFP (green) or tdTomato (red). (A) Whole mount images of ~80 day old organoid fusions show the emergence of GFP+ spots (arrows) in tdTomato+ tissue in ventral::dorsalUnt and ventral::dorsalCycA organoid fusions. (B) Tile-scan confocal images of immunostained mixed organoid fusion cryosections shows migration of GFP+ cells across the midline (dashed line) into the GFP- organoid. (C) Quantification of GFP+ cell density in GFP- tissue from tissue sections. Each data point corresponds to an individual organoid, and the data is represented as mean±SD with statistical significance tested using one-way ANOVA [F(3,19)=8.214, p=0.0010] with posthoc Tukey’s test for between group comparisons. The ventral::dorsalUnt (n=7 organoids) and ventral::dorsalCycA (n=8) fusions show the most migration of GFP+ cells compared to dorsalUnt::dorsalUnt (n=4) and dorsalCycA::dorsalCycA (n=4) fusions. Scale bars are 500µm.
Figure 4
Figure 4. GABAergic interneurons migrate between fused dorsal-ventral cerebral organoids.
(A) A whole organoid confocal tile-scan image of an immunostained 80-day old ventral::dorsalCycA organoid fusion cryosection. GFP+ cells can be observed migrating across the fusion midline (dashed line) from GFP+ ventral into GFP- dorsal tissue. The GABAergic marker GAD1 can be observed in a similar pattern as GFP (arrowheads). (B-C) A magnified view of peripheral (B) and internal regions (C) of the organoid fusion in (A). GFP+ cells expressing GAD1 can be observed in both regions (arrows). (D) A confocal image of GFP and HuC/D immunostaining in the dorsal region of an 80 day old ventral::dorsalCycA organoid fusion cryosection showing that migrating GFP+ cells express the neuronal marker HuC/D. Arrows point to cell bodies in focus in the single plane z-section. (E) A confocal image of GFP and NKX2-1 immunostaining in the dorsal region of an 80 day old ventral::dorsalCycA organoid fusion cryosection showing that some migrated GFP+ cells maintain NKX2-1 expression (arrows). (F) Quantification of the percentage (mean±SEM) of GFP+ migrated cells in dorsal organoid fusion tissue expressing HuC/D (100±0%, 1427 cells counted from n=4 organoids), GAD1 (57.5±3.0%, 1879 cells counted from n=4 organoids), or NKX2-1 (18.6±3.6%, 3067 cells counted from n=4 organoids). Scale bars are (A) 500µm, (B-E) 20µm.
Figure 5
Figure 5. Migrating interneurons in ventral::dorsal cerebral organoid fusions express various interneuron subtype markers.
(A-H) Confocal images of immunostaining in dorsal regions of 80-day old ventral::dorsalCycA organoid fusion cryosections. Expression of the GABAergic markers GAD1 or VGAT were used to identify interneurons. Examples of various migrated GFP+ interneurons expressing either GAD1 or VGAT were observed expressing the MGE-derived interneuron marker SOX6 (A) or subtype markers SOM (B), NPY (C), CB (D), and PV (E). Migrated GFP+ interneurons also expressed the LGE/CGE-derived interneuron markers SP8 (F), COUP-TFII (G), or the subtype marker CR (H). (I) Quantification of the percentage of GFP+/GAD1+ migrating interneurons in the dorsal region of organoid fusions expressing various subtype markers. SOX6 (38.7±3.9%, 1002 cells counted from n=4 organoids), PV (4.7±1.7%, 1879 cells counted from n=4 organoids), SST (6.4±1.5%, 818 cells counted from n=3 organoids), NPY (5.9±2.0%, 748 cells counted from n=5 organoids), CB (20.1±2.3%, 1114 cells counted from n=4 organoids), SP8 (33.0±5.2%, 620 cells counted from n=4 organoids). (J) Quantification of the percentage of GFP+/VGAT+ migrating interneurons in the dorsal region of organoid fusions expressing various subtype markers. CR (4.3±2.2%, 898 cells counted from n=4 organoids), COUPTFII (38.7±7.8%, 781 cells counted from n=4 organoids. Scale bars are 20µm. Abbreviations: SOM=somatostatin, NPY=neuropeptide Y, CB=calbindin, PV=parvalbumin, CR=calretinin, VGAT=vesicular GABA transporter, GAD1=glutamate decarboxylase 1.
Figure 6
Figure 6. Migrating cells in cerebral organoid fusions exhibit the migratory dynamics of tangentially migrating interneurons and are sensitive to CXCR4 activity.
(A) A schematic representation of the organoid fusion slice culture assay for either short-term time-lapse imaging of migratory dynamics, or long-term drug-treatments. A representative tile-scan image of an entire ventral::dorsalCycA organoid fusion slice is shown with the ventral GFP+ regions labeled in green and the unlabeled or tdTomato dorsal regions outlined in red. A region containing the migrating cell shown in (B) is noted by a yellow box. (B) Still images from a 3-day time-lapse experiment showing a migrating GFP+ cell. The branched leading process exhibits both extending (closed arrowheads) and retracting (open arrowheads) branches as the cell body follows one of the leading processes. (C) An extending neurite (closed arrowhead) with a tuft that appears to be an axon growth cone travels in one direction across the field of view. (D) A widefield image of GFP+ cells that migrated into the tdTomato+ dorsal region (red outline) from long-term organoid fusion slice cultures that were either untreated (control) or treated with a the CXCR4 inhibitor (AMD3100). (E) Quantification of the migrated GFP+ cell density with data represented as mean±SD with statistical significance tested using the student’s t-test (df=4) comparing control (n=3 organoids) to AMD3100 (n=3) treatment. Slices from the same organoid were split between control and AMD3100-treatments, and 3 different organoids were used from 2 different independent differentiations. Each data point represents cell density counts from one slice. Fewer migrating cells are observed in AMD3100-treated slice cultures. Scale bars are (A) 500µm, (B-C) 50µm, and (D) 500µm. The images in (A-C) are from an organoid fusion created by fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid. The images and data in (D-E) are from iPSC-derived organoid fusions.

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