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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Harris, K.D. & Shepherd, G.M.G. The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181 (2015).
Marín, O. & Müller, U. Lineage origins of GABAergic versus glutamatergic neurons in the neocortex. Curr. Opin. Neurobiol. 26, 132–141 (2014).
Kessaris, N., Magno, L., Rubin, A.N. & Oliveira, M.G. Genetic programs controlling cortical interneuron fate. Curr. Opin. Neurobiol. 26, 79–87 (2014).
Guo, J. & Anton, E.S. Decision making during interneuron migration in the developing cerebral cortex. Trends Cell Biol. 24, 342–351 (2014).
Marín, O., Valiente, M., Ge, X. & Tsai, L.H. Guiding neuronal cell migrations. Cold Spring Harb. Perspect. Biol. 2, a001834 (2010).
Marín, O. Interneuron dysfunction in psychiatric disorders. Nat. Rev. Neurosci. 13, 107–120 (2012).
Rossignol, E. Genetics and function of neocortical GABAergic interneurons in neurodevelopmental disorders. Neural Plast. 2011, 649325 (2011).
Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).
Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).
Renner, M. et al. Self-organized developmental patterning and differentiation in cerebral organoids. EMBO J. http://dx.doi.org/10.15252/embj.201694700 (2017).
Lancaster, M.A. & Knoblich, J.A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).
Maroof, A.M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).
Nicholas, C.R. et al. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 12, 573–586 (2013).
Liu, Y. et al. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells. Nat. Protoc. 8, 1670–1679 (2013).
Paşca, A.M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).
Kadoshima, T. et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc. Natl. Acad. Sci. USA 110, 20284–20289 (2013).
Martynoga, B., Morrison, H., Price, D.J. & Mason, J.O. Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis. Dev. Biol. 283, 113–127 (2004).
Xuan, S. et al. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 14, 1141–1152 (1995).
Hevner, R.F. et al. Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29, 353–366 (2001).
Cobos, I., Long, J.E., Thwin, M.T. & Rubenstein, J.L. Cellular patterns of transcription factor expression in developing cortical interneurons. Cereb. Cortex 16, i82–i88 (2006).
Eisenstat, D.D. et al. DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. J. Comp. Neurol. 414, 217–237 (1999).
Hernández-Miranda, L.R., Parnavelas, J.G. & Chiara, F. Molecules and mechanisms involved in the generation and migration of cortical interneurons. ASN Neuro 2, e00031 (2010).
Flames, N. et al. Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. J. Neurosci. 27, 9682–9695 (2007).
Butt, S.J.B. et al. The temporal and spatial origins of cortical interneurons predict their physiological subtype. Neuron 48, 591–604 (2005).
Xu, Q., Cobos, I., De La Cruz, E., Rubenstein, J.L. & Anderson, S.A. Origins of cortical interneuron subtypes. J. Neurosci. 24, 2612–2622 (2004).
Sussel, L., Marin, O., Kimura, S. & Rubenstein, J.L. Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126, 3359–3370 (1999).
Hsieh-Li, H.M. et al. Gsh-2, a murine homeobox gene expressed in the developing brain. Mech. Dev. 50, 177–186 (1995).
Georgala, P.A., Carr, C.B. & Price, D.J. The role of Pax6 in forebrain development. Dev. Neurobiol. 71, 690–709 (2011).
Vazin, T. et al. Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease. Neurobiol. Dis. 62, 62–72 (2014).
Erlander, M.G., Tillakaratne, N.J., Feldblum, S., Patel, N. & Tobin, A.J. Two genes encode distinct glutamate decarboxylases. Neuron 7, 91–100 (1991).
Meyer, G., Goffinet, A.M. & Fairén, A. What is a Cajal–Retzius cell? A reassessment of a classical cell type based on recent observations in the developing neocortex. Cereb. Cortex 9, 765–775 (1999).
Hevner, R.F., Neogi, T., Englund, C., Daza, R.A.M. & Fink, A. Cajal–Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin. Brain Res. Dev. Brain Res. 141, 39–53 (2003).
Abranches, E. et al. Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo. PLoS One 4, e6286 (2009).
Brown, J.P. et al. Transient expression of doublecortin during adult neurogenesis. J. Comp. Neurol. 467, 1–10 (2003).
Dehmelt, L. & Halpain, S. The MAP2/Tau family of microtubule-associated proteins. Genome Biol. 6, 204 (2005).
Nóbrega-Pereira, S. et al. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors. Neuron 59, 733–745 (2008).
Markram, H. et al. Interneurons of the neocortical inhibitory system. Nat. Rev. Neurosci. 5, 793–807 (2004).
Fishell, G. & Rudy, B. Mechanisms of inhibition within the telencephalon: “where the wild things are”. Annu. Rev. Neurosci. 34, 535–567 (2011).
Sultan, K.T., Brown, K.N. & Shi, S.-H. Production and organization of neocortical interneurons. Front. Cell. Neurosci. 7, 221–221 (2013).
Ma, T. et al. Subcortical origins of human and monkey neocortical interneurons. Nat. Neurosci. 16, 1588–1597 (2013).
Hansen, D.V. et al. Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences. Nat. Neurosci. 16, 1576–1587 (2013).
Tanaka, D.H., Maekawa, K., Yanagawa, Y., Obata, K. & Murakami, F. Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex. Development 133, 2167–2176 (2006).
Tanaka, D., Nakaya, Y., Yanagawa, Y., Obata, K. & Murakami, F. Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins. Development 130, 5803–5813 (2003).
Britto, J.M., Johnston, L.A. & Tan, S.-S. The stochastic search dynamics of interneuron migration. Biophys. J. 97, 699–709 (2009).
Ang, E.S.B.C. Jr., Haydar, T.F., Gluncic, V. & Rakic, P. Four-dimensional migratory coordinates of GABAergic interneurons in the developing mouse cortex. J. Neurosci. 23, 5805–5815 (2003).
Martini, F.J. et al. Biased selection of leading process branches mediates chemotaxis during tangential neuronal migration. Development 136, 41–50 (2009).
Tanaka, D.H. et al. Random walk behavior of migrating cortical interneurons in the marginal zone: time-lapse analysis in flat-mount cortex. J. Neurosci. 29, 1300–1311 (2009).
Reiner, O. LIS1 and DCX: implications for brain development and human disease in relation to microtubules. Scientifica (Cairo) 2013, 393975 (2013).
Fatehullah, A., Tan, S.H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).
Ranga, A., Gjorevski, N. & Lutolf, M.P. Drug discovery through stem cell-based organoid models. Adv. Drug Deliv. Rev. 69–70, 19–28 (2014).
Wang, Y. et al. CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron 69, 61–76 (2011).
Subramanian, L., Bershteyn, M., Paredes, M.F. & Kriegstein, A.R. Dynamic behaviour of human neuroepithelial cells in the developing forebrain. Nat. Commun. 8, 14167 (2017).
Letinic, K., Zoncu, R. & Rakic, P. Origin of GABAergic neurons in the human neocortex. Nature 417, 645–649 (2002).
Jakovcevski, I., Mayer, N. & Zecevic, N. Multiple origins of human neocortical interneurons are supported by distinct expression of transcription factors. Cereb. Cortex 21, 1771–1782 (2011).
Yu, X. & Zecevic, N. Dorsal radial glial cells have the potential to generate cortical interneurons in human but not in mouse brain. J. Neurosci. 31, 2413–2420 (2011).
Stumm, R.K. et al. CXCR4 regulates interneuron migration in the developing neocortex. J. Neurosci. 23, 5123–5130 (2003).
Lewis, D.A. Inhibitory neurons in human cortical circuits: substrate for cognitive dysfunction in schizophrenia. Curr. Opin. Neurobiol. 26, 22–26 (2014).
Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 455, 54–59 (2017).
Nakano, T. et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785 (2012).
Sakaguchi, H. et al. Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue. Nat. Commun. 6, 8896 (2015).
Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Reports 10, 537–550 (2015).
Jo, J. et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 19, 248–257 (2016).
Bagley, J.A., Reumann, D. Knoblich J.A. Detailed cerebral organoid fusion method. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.064 (2017).
Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).
Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463–1465 (2009).
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).
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.
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.
(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.
(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 Figures 1-6 and Supplementary Tables 1-3
Detailed Cerebral Organoid Fusion Method
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.
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.
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.
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.
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.
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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