Fused cerebral organoids model interactions between brain regions

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.

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Figure 1: Patterning ventral-forebrain-containing cerebral organoids.
Figure 2: Fused cerebral organoids as a model for cell migration.
Figure 3: Robust migration from ventral into dorsal regions of fused cerebral organoids.
Figure 4: GABAergic interneurons migrate between fused dorsal–ventral cerebral organoids.
Figure 5: Migrating interneurons in ventral::dorsal cerebral organoid fusions express various interneuron subtype markers.
Figure 6: A system to study the dynamics of migrating cells in cerebral organoid fusions.

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Acknowledgements

We are grateful to members of the Knoblich laboratory for technical expertise and feedback. We also thank the Molecular Biology Service Facility, notably H. Scheuch; the BioOptics facility, notably T. Müller and P. Pasierbek; and all the core facilities of IMBA/IMP for technical support. We also thank the HistoPathology facility of the Vienna Biocenter Core Facilities (VBCF). We also thank E.H. Gustafson and S. Wolfinger for technical assistance and expertise regarding cell culture and 3D cerebral organoid culture. J.A.B. received funding from an EMBO postdoctoral fellowship. Work in J.A.K.'s laboratory is supported by the Austrian Academy of Sciences, the Austrian Science Fund (Z_153_B09), and an advanced grant from the European Research Council (ERC).

Author information

J.A.B. and J.A.K. conceived the project and experimental design and wrote the manuscript. J.A.B. performed experiments and analyzed data. D.R. performed experiments and analyzed data under the supervision of J.A.B. and J.A.K. S.B. and J.L.-S. performed cell counting of immunostained tissue. J.A.K. directed and supervised the project.

Correspondence to Juergen A Knoblich.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ventral drug-patterning treatment induces ventral forebrain identity in cerebral organoids.

(A-F) Whole-organoid confocal tile scan images of dorsalUnt and ventral organoids (48-55 days old) immunostained for forebrain (FOXG1), dorsal (TBR1, PAX6), or ventral (NKX2-1, DLX2, or GSX2) markers. (G) Quantification of the percentages (mean±SEM) of VZ-like regions expressing each marker: FOXG1 (dorsalUnt 82.5±9.1%, ventral 82.5±4.2%, p>0.999), NKX2-1 (dorsalUnt 0.0±0.0%, ventral 73.1±7.5%, p<0.001), TBR1 (dorsalUnt 95.5±3.6%, ventral 0.0±0.0%, p<0.001), DLX2 (dorsalUnt 4.75±2.3%, ventral 96.8±1.4%, p<0.001), PAX6 (dorsalUnt 76.3±11.2%, ventral 0.0±0.0%, p<0.001), GSX2 (dorsalUnt 6.0±2.0%, ventral 100.0±0.0%, p<0.001). For each marker, 4 organoids were used for counting. Statistical significance was tested using the student's t-test (df=6). Scale bars are 500μm.

Supplementary Figure 2 The gross morphology of cerebral organoid fusions changes with age.

Whole-organoid confocal tile-scans to visualize the gross structural organization of ventral::dorsalCycA cerebral organoid fusion tissue at different ages. (A) 46 day old and (B) 61 day old organoid fusions contained VZ-like progenitor regions (insets A and B). Older, 80 day old organoid fusions contained less or no VZ-like progenitor regions. Scale bars are 500μm.

Supplementary Figure 3 Migrating GFP+ cells in organoid fusions are highly non-proliferative.

(A) Confocal images showing GFP/Ki67 immunostaining of migrated GFP+ cells in the dorsal region of 46 and 80 day old ventral::dorsalCycA organoid fusion cryosections. Very few GFP+ cells also express Ki67 (yellow arrows). (B) Quantification of the percentage (mean±SEM) of GFP+ migrated cells expressing Ki67 from 46 day old (1.1±0.2%, 2420 cells counted from n=4 organoids), and 80 day old ventral::dorsalCycA fusions (0.7±0.2%, 3067 cells counted from n=4 organoids). Scale bar is 20μm.

Supplementary Figure 4 Migrating GFP+ cells in organoid fusions do not express the Cajal Retzius cell marker Reelin (RELN).

(A) A confocal image of GFP/RELN immunostaining in the dorsal region of an 80-day old ventral::dorsalCycA organoid fusion cryosection showing that migrated GFP+ cells (arrows) do not express RELN. Scale bar is 20μm.

Supplementary Figure 5 Migrating GFP+ cells in organoid fusions express immature and mature neuronal markers.

(A) A confocal image of GFP/DCX/NeuN immunostaining in the dorsal region of a 58-day old ventral::dorsalCycA organoid fusion cryosection showing that migrating GFP+ cells are DCX+ immature neurons (yellow arrows), and some are mature (DCX+/NeuN+) neurons (blue arrows). (B) A confocal image of GFP/MAP2 immunostaining in the dorsal region of an 80-day old ventral::dorsalCycA organoid fusion cryosection showing that some migrating GFP+ are mature (MAP2+) neurons (yellow arrows). Scale bars are 20μm.

Supplementary Figure 6 The morphology of GFP+ cells migrating within cerebral organoid fusions.

(A-C) Cropped z-projections of 80x spinning disc z-stacks to visualize the morphology of single GFP+ cells that migrated from ventral into dorsal organoid tissue within 80 day old ventral::dorsalCycA cerebral organoid fusions. (A) A GFP+GAD1+ interneuron with a branched morphology. The branches extend in many directions, and the cell body is large and round. (B-C) GFP+/GAD1+ interneurons with a migratory morphology consisting of an elongated cell body as well as branched leading processes and a trailing process. The cell in C has a leading process with 3 branches, and a bifurcated trailing process. Scale bars are 10μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-6 and Supplementary Tables 1-3

Supplementary Protocol

Detailed Cerebral Organoid Fusion Method

Supplementary Video 1

A cell migrating in cerebral organoid fusions, example 1. A time-lapse movie of migrating GFP+ cells within the dorsal region of a ventral/GFP::dorsalCycA organoid fusion. The cell migrates in a single direction. The leading process is branched with the different branches dynamically extending and retracting seemingly independent of one another. The trailing process follows as the cell body moves forward, and multiple times a leading process becomes a trailing process. As the cell moves forward, one leading process is extended while the remaining processes retract. Then the whole migratory dynamic cycle is repeated as the cell progresses forward. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid.

Supplementary Video 2

A cell migrating in cerebral organoid fusions, example 2. A time-lapse movie of migrating GFP+ cells within the dorsal region of a ventral/GFP::dorsalCycA organoid fusion. This movie is an example of a cell exhibiting many changes of direction involving the dynamic extension and retracting of several processes. As the cell body remains static, branches are extended in multiple directions, and then each of the main branches extends additional higher order branches. Finally, a branch is extended in a particular direction followed by the retraction of the other main branch. The cell body is then moved in the direction of the extending branch. The cycle is repeated as the cell decides which direction to migrate. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycAiPSC-derived organoid.

Supplementary Video 3

A cell migrating in cerebral organoid fusions, example 3. A time-lapse movie of migrating GFP+ cells within the dorsal region of a ventral/GFP::dorsalCycA organoid fusion. This movie shows multiple migrating cells. 1) Initially a cell in the middle of the field of view is migrating upward. The upward process is retracted as a new leading process is extended downward and becomes branched. The cell migration direction is then changed downward. The bifurcated leading process is dynamic such that one process is extended as the other process is retracted. The cell body then moves toward the extended leading process. Prior to nucleokinesis, a swelling is observed moving from the cell body into the proximal portion of the leading process. Then the cell body is moved in parallel to the swelling, and finally the cell body moves into the swelling. 2) A second cell migrates from the left field of view toward the right, changes direction back toward the left, and then again changes direction toward the right, and finally changes once again back toward the left. With each change of direction, the trailing process becomes the leading process. The new leading process is extended toward the direction of travel as the trailing process is retracted. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid.

Supplementary Video 4

A cell migrating in cerebral organoid fusions, example 4. A time-lapse movie of migrating GFP+ cells within the dorsal region of a ventral/GFP::dorsalCycA organoid fusion. This movie shows multiple cells migrating in different directions with extending and retracting processes. Beginning around 45 hours, one cell migrates throughout the entire field of view beginning in the top left corner and traveling toward the bottom right corner. The cell travels rapidly in a constant direction, but at around 70 hours the progress is slowed as the leading process branches. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid.

Supplementary Video 5

A cell migrating in cerebral organoid fusions, example 5. A time-lapse movie of migrating GFP+ cells within the dorsal region of a ventral::dorsalCycA organoid fusion. This movie shows multiple cells migrating. Around 6 hours a cell can be seen migrating into view from the bottom left corner of the field of view. This cell initially migrates left to right with a branched leading process. At multiple times, 3 branches are observed. As the cell progresses forward, branches are extended in the direction of travel, while other branches are retracted. Around 23 hours the cell changes direction abruptly from moving right to moving left. This involves an extension of a new process toward the left, while the previous leading process oriented to the right is retracted. The cell proceeds to the left, but around 39 hours, the leading process begins turning toward the right. The leading process makes a 180-degree turn, and then extends. The cell body then follows the leading process as the cell migrates rapidly from top to bottom and eventually proceeds out of view in the z-direction. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid.

Supplementary Video 6

Neurite dynamics within organoid fusions resembling growing axons. A time-lapse movie of ventral-derived GFP+ neurites growing within the dorsal region of a ventral/GFP::dorsalCycA organoid fusion. The neurites appear to be axons with an enlarged tuft at the end the processes which resembles that of a growth cone. The processes are highly dynamic, and exhibit extension in single directions, but also abrupt changes in direction. This recording was from a slice culture of an organoid fusion created fusing a ventral H9 hESC-derived organoid containing a CAG-eGFP-WPRE construct to a dorsalCycA iPSC-derived organoid.

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Bagley, J., Reumann, D., Bian, S. et al. Fused cerebral organoids model interactions between brain regions. Nat Methods 14, 743–751 (2017). https://doi.org/10.1038/nmeth.4304

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