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Cited by 788Pluripotent Stem Cells ResearchNeurogenesis and neuroplasticity mechanismsDevelopmental Biology and Gene Regulation

The use of brain organoids to investigate neural development and disease

Open access
Di Lullo, Elizabeth, Kriegstein, Arnold R.·2017·Nature Reviews Neuroscience
DOI 10.1038/nrn.2017.107PMC PMC5667942
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Abstract

Understanding the development and dysfunction of the human brain is a major goal of neurobiology. Much of our current understanding of human brain development has been derived from the examination of post-mortem and pathological specimens, bolstered by observations of developing non-human primates and experimental studies focused largely on mouse models. However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that enable the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of the development of the human brain and enable a detailed study of the pathogenesis of inherited and acquired brain diseases.

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Crossref · 10.1038/nrn.2017.107

Cites

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Claims the paper makes

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  • Findingcitation

    Production of radial glial cells, intermediate progenitors, and deep- and superficial-layer neurons in an ordered temporal fashion has been reported by studies using all current protocols.

    “The production of radial glial cells, intermediate progenitors, and deep- and superficial-layer neurons in an ordered temporal fashion has been reported by studies using all of the protocols currently in use2,3,5,25.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [2] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). This was the first paper to introduce a method for generating cerebral organoids. The organoids were used to model microcephaly. source

      Not checked in the free run.

    • cites [3] 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). This study provided a detailed exploration of brain organoid tissue including the temporal and spatial organization of cell type diversity and patterning. source

      Not checked in the free run.

    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

    • cites [25] Paşca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015). source

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  • Findingcitation

    Protocols have been published for organoids modeling the hippocampus, midbrain, hypothalamus, cerebellum, anterior pituitary, and retina.

    “However, protocols have also been published for the generation of organoids that model the development of other human brain regions, including the hippocampus7, midbrain4,5,8, hypothalamus5, cerebellum5,9, anterior pituitary10 and retina10,11.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [7] 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). source

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    • cites [4] 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). source

      Not checked in the free run.

    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

    • cites [8] Monzel, A. S. et al. Derivation of human midbrain-specific organoids from neuroepithelial stem cells. Stem Cell Rep. 8, 1144–1154 (2017). source

      Not checked in the free run.

    • cites [9] 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 Rep. 10, 537–550 (2015). source

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  • Findingreasoning

    Most protocols optimize neural induction by mimicking endogenous patterning through exogenous cues.

    “Most protocols therefore optimize neural induction by mimicking endogenous patterning through the application of exogenous cues (TABLE 1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites TABLE 1

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  • Findingnone given

    Several key features of in vivo brain organogenesis are recapitulated by in vitro organoids.

    “Nevertheless, several key features of in vivo brain organogenesis are recapitulated by in vitro organoids, making them attractive models for studies of certain aspects of brain development.”
    Quote in the paperSubstantial claim
  • Findingcitation

    Protocols using medium free of neural induction signalling molecules promote generation of diverse brain regions and cell populations.

    “Some protocols build on the principles of spontaneous neural induction by using medium that is free of neural induction signalling molecules2,27. This promotes the generation of diverse brain regions and cell populations2,28,29.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [2] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). This was the first paper to introduce a method for generating cerebral organoids. The organoids were used to model microcephaly. source

      Not checked in the free run.

    • cites [27] Lindborg, B. A. et al. Rapid induction of cerebral organoids from human induced pluripotent stem cells using a chemically defined hydrogel and defined cell culture medium. Stem Cells Transl Med. 5, 970–979 (2016). source

      Not checked in the free run.

    • cites [28] Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017). This study analysed gene expression in over 80,000 individual cells and found that organoids can generate a broad diversity of cells that are related to endogenous classes, including cells from the cerebral cortex and the retina. Neuronal activity within organoids was also controlled using light stimulation of photosensitive cells. source

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    • cites [29] Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl Acad. Sci. USA 112, 15672–15677 (2015). source

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  • May combine two claims. 36
  • Classified as probably not a substantial claim. 5

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    Findingcitation

    Such regional diversity leads to relatively variable outcomes and differentiation of some cells into non-ectodermal cell types.

    “Although such regional diversity is appealing, it also leads to a relatively variable outcome and the differentiation of some cells into non-ectodermal cell types28,29.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [28] Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017). This study analysed gene expression in over 80,000 individual cells and found that organoids can generate a broad diversity of cells that are related to endogenous classes, including cells from the cerebral cortex and the retina. Neuronal activity within organoids was also controlled using light stimulation of photosensitive cells. source

      Not checked in the free run.

    • cites [29] Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl Acad. Sci. USA 112, 15672–15677 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    WNT3A or a WNT pathway activator used with SMAD inhibition at early stages of forebrain organoid protocols promoted neuroepithelial-like organoids and significantly reduced cell death.

    “WNT3A or a WNT pathway activator has therefore been used in combination with SMAD inhibition at early stages of forebrain organoid protocols, where it was found to promote production of neuroepithelial-like organoids and significantly reduce cell death5.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Inhibition of WNT signalling promotes anterior identity and enhances neurectodermal differentiation during early embryoid body differentiation, whereas activation posteriorizes the embryoid body and promotes mesoderm differentiation.

    “Similar to its in vivo patterning, inhibition of WNT signalling promotes anterior identity and enhances neurectodermal differentiation during early-stage embryoid body differentiation, whereas activation of WNT signalling posteriorizes the embryoid body and promotes mesoderm differentiation34.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] ten Berge, D. et al. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–518 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    The developmental timing of cortical neurogenesis appears conserved in vitro and has been demonstrated with both suspension and adherent differentiation methods.

    “Interestingly, the developmental timing of cortical neurogenesis appears to be conserved in vitro and has been demonstrated with both suspension and adherent differentiation methods2,3,5,39.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [2] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). This was the first paper to introduce a method for generating cerebral organoids. The organoids were used to model microcephaly. source

      Not checked in the free run.

    • cites [3] 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). This study provided a detailed exploration of brain organoid tissue including the temporal and spatial organization of cell type diversity and patterning. source

      Not checked in the free run.

    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

    • cites [39] Otani, T., Marchetto, M. C., Gage, F. H., Simons, B. D. & Livesey, F. J. 2D and 3D stem cell models of primate cortical development identify species-specific differences in progenitor behavior contributing to brain size. Cell Stem Cell 18, 467–480 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Some cell types were only produced by a subset of organoids, and organoids grown in the same bioreactor tended to be similar, suggesting that batch effects and local microenvironments are important in patterning.

    “Also, the authors observed that some cell types were only produced by a subset of organoids and that organoids grown in the same bioreactor tended to be similar in terms of their ability to make cells of each cluster, suggesting that batch effects and local microenvironments are important in patterning and possibly that organoids secrete signalling factors that influence sister organoids.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017). This study analysed gene expression in over 80,000 individual cells and found that organoids can generate a broad diversity of cells that are related to endogenous classes, including cells from the cerebral cortex and the retina. Neuronal activity within organoids was also controlled using light stimulation of photosensitive cells. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    DNA methylation analysis in brain organoids revealed that some patterns were conserved between fetal tissue and organoids, whereas others were specific to in vitro conditions.

    “A recent study analysed DNA methylation in brain organoids and revealed that, although some patterns were conserved between fetal tissue and brain organoids, others were specific to in vitro conditions52.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Luo, C. et al. Cerebral organoids recapitulate epigenomic signatures of the human fetal brain. Cell Rep. 17, 3369–3384 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    6-month-old organoids presented greater diversity of cell types than 3-month-old organoids, as well as enrichment of genes associated with neuronal and glial maturation.

    “As one might expect, 6-month-old organoids presented a greater diversity of cell types than 3-month-old organoids, as well as enrichment of genes associated with neuronal and glial maturation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017). This study analysed gene expression in over 80,000 individual cells and found that organoids can generate a broad diversity of cells that are related to endogenous classes, including cells from the cerebral cortex and the retina. Neuronal activity within organoids was also controlled using light stimulation of photosensitive cells. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    A large number of cells were assigned to a mesodermal lineage, emphasizing variability inherent in the protocol.

    “For example, a large number of cells were assigned to a mesodermal lineage, emphasizing the variability inherent in the protocol.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017). This study analysed gene expression in over 80,000 individual cells and found that organoids can generate a broad diversity of cells that are related to endogenous classes, including cells from the cerebral cortex and the retina. Neuronal activity within organoids was also controlled using light stimulation of photosensitive cells. source

      Not checked in the free run.

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    Findingcitation

    These distinct neuronal subclasses exhibited multilaminar organization, although they did not form the six distinct layers seen in normal mammalian cortex.

    “Remarkably, these distinct subclasses of neuronal cells exhibited multilaminar organization, although they did not form the six distinct layers seen in normal mammalian cortex (FIG. 2a).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Migrated cells fired action potentials at twice the rate of cells in unfused subpallial spheres or non-migrated cells in the fused organoid, suggesting maturation of membrane properties.

    “The cells that had migrated into the cortical spheroids also fired action potentials at twice the rate of cells in unfused subpallial spheres or non-migrated cells in the fused organoid, suggesting maturation of membrane properties.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017). This study used pre-patterned pallial and subpallial spheroids to form fused forebrain spheroids, demonstrating interneuron diversity, migration and integration. This organoid model was used to model Timothy syndrome. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    In the dorsal-ventral fused model, cells that migrated from the subpallial spheroid to the cortical spheroid expressed cortical interneuron markers, and whole-cell voltage-clamp recordings showed that these interneuron-like cells can receive excitatory inputs from dorsal cortical organoid cells and that dorsal cortical cells can receive inputs from inhibitory interneurons.

    “In the dorsal-ventral fused model mentioned earlier6, cells that migrated from the subpallial spheroid to the cortical spheroid expressed cortical interneuron markers, and whole-cell voltage-clamp recordings demonstrated that these interneuron-like cells can receive excitatory inputs from cells in the dorsal cortical organoids; conversely, the cells in the dorsal cortical organoids can receive”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017). This study used pre-patterned pallial and subpallial spheroids to form fused forebrain spheroids, demonstrating interneuron diversity, migration and integration. This organoid model was used to model Timothy syndrome. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Neurons fired action potentials spontaneously, there was evidence of synaptogenesis, and excitatory postsynaptic potentials were evoked by extracellular electrical stimulation, suggesting neuronal connectivity.

    “Neurons fired action potentials spontaneously, there was evidence of synaptogenesis and excitatory postsynaptic potentials were evoked in response to extracellular electrical stimulation, suggesting neuronal connectivity.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Paşca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    WNT signalling effects are stage specific, with divergent roles in cell proliferation and cell type specification depending on temporal and regional expression.

    “For example, in embryogenesis, the effects of WNT signalling are stage specific, and there are divergent roles for WNT signalling in cell proliferation and cell type specification that depend on its temporal and regional expression (for reviews, see REFS 30,31).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [30] Suzuki, I. K. & Vanderhaeghen, P. Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells. Development 142, 3138–3150 (2015). source

      Not checked in the free run.

    • cites [31] Harrison-Uy, S. J. & Pleasure, S. J. Wnt signaling and forebrain development. Cold Spring Harb. Perspect. Biol. 4, a008094 (2012).

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    All organoids undergo some extent of self-organization, but the mechanisms of self-organization are not well understood.

    “Despite differences in protocols, all organoids undergo some extent of self-organization; however, the mechanisms of self- organization are not well understood.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A major limitation of organoid modelling is the lack of evidence for faithful cortical circuits, although some progress has been reported.

    “A major limitation of organoid modelling is the lack of evidence of the establishment of faithful cortical circuits; however, some progress has been reported on this front.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    Organoids show structural features of ventricular zones and some aspects of cortical layering, but additional levels of cortical organization such as the radial glial scaffold, gyrification, proper cortical layering, and specificity of neuronal connections remain to be established.

    “Organoids show structural features of ventricular zones and some aspects of cortical layering (FIG. 2a), but additional levels of cortical organization such as the radial glial scaffold, gyrification, proper cortical layering and the specificity of neuronal connections remain to be established and need to be compared to patterns observed in primary tissue (FIG. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Cerebral organoids grown from iPSCs of a microcephalic patient carrying a CDK5RAP2 mutation had fewer proliferating progenitor cells and showed premature neural differentiation compared with wild-type cells.

    “Cerebral organoids grown from these cells contained fewer proliferating progenitor cells and showed premature neural differentiation compared to wild-type counterparts.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). This was the first paper to introduce a method for generating cerebral organoids. The organoids were used to model microcephaly. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    oRGs are present in large numbers in gyrencephalic primates and are considered pivotal to the evolutionary increase in human cortex size and complexity.

    “oRGs are present in large numbers in gyrencephalic primates22,23,40–44 and are considered pivotal to the evolutionary increase in human cortex size and complexity45,46.”
    Quote in the paperNot a substantial claim0 of 9 sources checked
    • cites [22] Fietz, S. A. et al. OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling. Nat. Neurosci. 13, 690–699 (2010). source

      Not checked in the free run.

    • cites [23] Hansen, D. V., Lui, J. H., Parker, P. R. L. & Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010). source

      Not checked in the free run.

    • cites [40] Smart, I. H. M., Dehay, C., Giroud, P., Berland, M. & Kennedy, H. Unique morphological features of the proliferative zones and postmitotic compartments of the neural epithelium giving rise to striate and extrastriate cortex in the monkey. Cereb. Cortex 12, 37–53 (2002). source

      Not checked in the free run.

    • cites [41] Lukaszewicz, A. et al. G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex. Neuron 47, 353–364 (2005). source

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    • cites [42] Zecevic, N., Chen, Y. & Filipovic, R. Contributions of cortical subventricular zone to the development of the human cerebral cortex. J. Comp. Neurol. 491, 109–122 (2005). source

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    • cites [43] Dehay, C. & Kennedy, H. Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450 (2007). source

      Not checked in the free run.

    • cites [44] Martínez- Cerdeño, V. et al. Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents. PLoS ONE 7, e30178 (2012). source

      Not checked in the free run.

    • cites [45] Lui, J. H., Hansen, D. V. & Kriegstein, A. R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011). source

      Not checked in the free run.

    • cites [46] Fish, J. L., Dehay, C., Kennedy, H. & Huttner, W. B. Making bigger brains-the evolution of neural-progenitor-cell division. J. Cell Sci. 121, 2783–2793 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    This suggests that hem-like signalling centres can participate in the self-organization of cerebral organoids, mimicking endogenous patterning of the developing brain.

    “This suggests that hem-like signalling centres can participate in the self-organization of cerebral organoids, mimicking the endogenous patterning of the developing brain.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    The brain organoid model is at an early phase of development.

    “The brain organoid model is in an early phase of development.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Both organoid and primary tissue studies found high infectivity of neural progenitor cells, but astrocyte infection was only occasionally observed in organoids while it was abundant in primary tissue.

    “Studies using organoids and primary tissue both found high infectivity of NPCs; however, infection of astrocytes was only occasionally observed in organoids5 but was abundantly present in primary tissue72.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

    • cites [72] Retallack, H. et al. Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proc. Natl Acad. Sci. USA 113, 14408–14413 (2016). source

      Not checked in the free run.

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    Premisecitation

    AXL is a crucial receptor for infection of human glial cells (astrocytes and microglia) but not of human neural progenitor cells.

    “another study reporting that AXL is a crucial receptor for infection of human glial cells (astrocytes and microglia) but not of human NPCs75.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [75] Meertens, L. et al. Axl mediates ZIKA virus entry in human glial cells and modulates innate immune responses. Cell Rep. 18, 324–333 (2017). source

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    Premisecitation

    Single-cell RNA-seq of human primary tissue showed that the candidate viral entry receptor AXL is highly expressed by cortical radial glial cells, astrocytes, endothelial cells and microglia, and also by radial-glia-like cells of brain organoids.

    “single-cell RNA-seq data from human primary tissue revealed that the candidate viral entry receptor, AXL (also known as tyrosine-protein kinase receptor UFO), is highly expressed by human cortical radial glial cells, astrocytes, endothelial cells, and microglia, as well as in radial-glia-like cells of brain organoids73.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [73] Nowakowski, T. J. et al. Expression analysis highlights AXL as a candidate zika virus entry receptor in neural stem cells. Cell Stem Cell 18, 591–596 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Blocking the AXL receptor reduced Zika virus infection of PSC-derived astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection.

    “Blocking the AXL receptor reduced ZIKV infection of PSC-derived astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [73] Nowakowski, T. J. et al. Expression analysis highlights AXL as a candidate zika virus entry receptor in neural stem cells. Cell Stem Cell 18, 591–596 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The importance of AXL and the greater susceptibility of astrocytes and microglia to Zika virus could easily have been missed using brain organoid models alone, suggesting that more sophisticated organoids may be needed for some aspects of disease modelling and drug discovery.

    “The importance of AXL (or other receptors and pathways implicated in glial ZIKV infection) and the enhanced susceptibility of astrocytes and microglia to infection could have easily been overlooked based on brain organoid models alone, suggesting that more sophisticated organoids may be required for some aspects of disease modelling and drug discovery.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Pathway analysis of gene expression changes during TLR3 activation highlighted genes related to neuronal development, suggesting a mechanistic link to disrupted neurogenesis.

    “Pathway analysis of gene expression changes during TLR3 activation highlighted several genes related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [71] Dang, J. et al. Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell 19, 258–265 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A second report observed altered division mode of radial glial cells in Miller-Dieker syndrome organoids and identified non-cell-autonomous WNT signalling defects as an underlying mechanism.

    “A second report also observed changes in the division mode of radial glial cells in MDS organoids and identified non-cell-autonomous defects in WNT signalling as an underlying mechanism67.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [67] Iefremova, V. et al. An organoid-based model of cortical development identifies non-cell-autonomous defects in wnt signaling contributing to Miller–Dieker syndrome. Cell Rep. 19, 50–59 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    2D cell cultures may not provide the complex extracellular environment needed to model extracellular protein aggregation, making 3D tissue-like structures an arguably more promising model.

    “2D cell cultures may not provide the complex extracellular environment necessary to model extracellular protein aggregation, making 3D tissue-like structures an arguably more promising model.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Not all aspects of brain structure and function will be modelled by organoids, but the earliest events in disease aetiology may be captured and may share mechanistic pathways with features that appear at later stages.

    “Of course, not all aspects of brain structure and function will be modelled by these tissue structures, but perhaps the most precocious events in disease aetiology can be captured and investigated, and these may share mechanistic pathways with disease features that manifest at later stages.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationnone given

    The absence of cell types involved in normal brain development and circuit function, variation in structural features of cell and tissue architecture, and the need for more comprehensive transcriptional network matching between organoid and fetal tissue must be overcome to realize the full potential of these systems as models of human brain development and disease.

    “the absence of cell types involved with normal brain development and circuit function, the variation in structural features of cell and tissue architecture, and the need for more comprehensive transcriptional network matching between organoid and fetal tissue will need to be overcome to realize the full potential of these systems as models for human brain”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Forebrain organoid studies revealed cell-specific viral tropism, selective effects on neural progenitor cell proliferation, and upregulation of the innate immune receptor TLR3 after Zika virus infection.

    “Further studies used forebrain organoids to reveal cell-specific viral tropism, selective effects on NPC proliferation5, and upregulation of the innate immune receptor Toll-like receptor 3 (TLR3) after ZIKV infection71.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016). This study used mini bioreactors to produce forebrain organoids with a well-defined OSVZ and demonstrated the presence of oRGs with defined molecular markers. Diverse neuronal cell types expressing molecular markers of all six cortical layers were observed. source

      Not checked in the free run.

    • cites [71] Dang, J. et al. Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell 19, 258–265 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The amyloid hypothesis of Alzheimer disease holds that excessive accumulation of amyloid-beta peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau.

    “The amyloid hypothesis of AD posits that excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau84.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [84] Serrano-Pozo, A., Frosch, M. P., Masliah, E. & Hyman, B. T. Neuropathological alterations in Alzheimer disease. Cold Spring Harb. Perspect. Med. 1, a006189 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationreasoning

    The failure of many neurotherapeutic approaches to translate from animal models to clinical practice underscores the need for better predictive models, and brain organoids may help bridge this divide.

    “The failure of many neurotherapeutic approaches to translate from animal models to clinical practice underscores the need for better predictive models, and brain organoids may help bridge this divide.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    Brain organoids are amenable to drug treatment, and Alzheimer disease phenotypes can be recapitulated across multiple cell lines derived from different patients.

    “Brain organoids are amenable to drug treatment, and it has been shown that disease phenotypes can be recapitulated across multiple cell lines derived from different patients with AD.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Forebrain organoids derived from four patients with severe idiopathic autism spectrum disorder showed dysregulation of FOXG1 expression, accelerated cell cycle progression and increased production of GABAergic neurons.

    “Patient-derived organoids exhibited dysregulation of forkhead box G1 expression (an important gene for fore-brain development), accelerated cell cycle progression and increased production of GABAergic neurons.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [83] Mariani, J. et al. FOXG1-Dependent dysregulation of GABA/Glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    2D iPSC models have been important for analysing gene expression, cell morphology, neuronal excitability and synapse formation, and have shed light on mechanisms underlying schizophrenia, bipolar disorder and Rett syndrome.

    “2D iPSC models have been important for analysing parameters such as gene expression, cell morphology, neuronal excitability, and synapse formation76,77 and have shed light on mechanisms underlying disorders such as schizophrenia78,79, bipolar disorder78–80, and Rett syndrome81”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [76] Wen, Z., Christian, K. M., Song, H. & Ming, G.-L. Modeling psychiatric disorders with patient-derived iPSCs. Curr. Opin. Neurobiol. 36, 118–127 (2016). source

      Not checked in the free run.

    • cites [77] Flaherty, E. K. & Brennand, K. J. Using hiPSCs to model neuropsychiatric copy number variations (CNVs) has potential to reveal underlying disease mechanisms. Brain Res. 1655, 283–293 (2017). source

      Not checked in the free run.

    • cites [78] Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221–225 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Miller-Dieker syndrome organoids also showed severe apoptosis of neuroepithelial stem cells in the ventricular-like zone and a mitotic defect in outer radial glia.

    “The organoids also displayed severe apoptosis of neuroepithelial stem cells in the ventricular-like zone and a mitotic defect in oRGs50.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [50] Bershteyn, M. et al. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell 20, 435–449.e4 (2017). This study used brain organoids to model lissencephaly. The authors identified a mitotic defect in oRGs and used single-cell RNA sequencing to identify cells presenting an oRG transcriptional signature in human brain organoids. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    The slow maturation of organoids may limit studies of later developmental events or stages of disease expression, whereas disease phenotypes that manifest at early stages may be aptly modelled.

    “The slow maturation of organoids may limit studies of later developmental events or stages of disease expression; however, disease phenotypes that manifest at early stages may be aptly modelled.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Brain organoids can also be used to uncover human-specific phenotypes, which gives them an advantage over existing mouse models.

    “However, they can also be used to discover human-specific phenotypes, providing an advantage over existing mouse models.”
    Quote in the paperSubstantial claim
39 of 39

  • The paper relies on this without support.

    Existing tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development.

    “However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development.”

  • The paper relies on this without support.

    Organoid generation relies on the ability of stem and progenitor cells to self-organize to form complex tissue structures.

    “Organoid generation relies on the remarkable ability of stem and progenitor cells to self-organize to form complex tissue structures.”

  • The paper relies on this without support.

    Most human cortical circuits develop postnatally, and many take years to mature.

    “Most human cortical circuits develop postnatally, and many take years to mature.”

  • The paper relies on this without support.

    Animal models of neurodevelopmental diseases have species differences that limit their resemblance to human diseases.

    “Although animal models of neurodevelopmental diseases have been essential for our current understanding of pathological mechanisms, there are obvious species differences that limit their resemblance to human diseases, including differences in developmental programmes, cytoarchitecture, cell composition, and genetic background.”

  • The paper relies on this without support.

    Many uniquely human cognitive and behavioural diseases present polygenic aetiology, making them difficult to study with existing animal models.

    “However, many uniquely human cognitive and behavioural diseases — such as autism spectrum disorder (ASD) or schizophrenia — present polygenic aetiology, making them difficult to study with existing animal models.”

  • The paper relies on this without support.

    Many neuropsychiatric diseases involve defects in processes that unfold after birth, such as circuit formation, synaptic pruning, dendritic growth and cortical circuit refinement.

    “Many neuropsychiatric diseases manifest defects in processes that unfold postnatally, such as circuit formation, synaptic pruning, dendritic growth and cortical circuit refinement.”

  • The paper relies on this without support.

    Human neuronal networks take years to mature and depend on subcortical influences.

    “These neuronal networks can take years to mature in the human brain and rely on subcortical influences”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • PSC-based models have remained difficult to use to study cortical neuron development in vitro, given the technical challenges of long-term maintenance of functional neurons, whether as monolayer cultures or in organoids.

    “they have remained difficult to use to study cortical neuron development in vitro, given the technical challenges of long-term maintenance of functional neurons, whether as monolayer cultures or in organoids”

    From Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits (2019) · cites this paper as Di Lullo and Kriegstein, 2017

    The Challenges and Establishing functional cortical circuits sections describe organoid immaturity and the difficulty of developing faithful, mature cortical circuits. The Generating organoids section mentions monolayer protocols for producing cortical neurons, but does not identify long-term maintenance of functional neurons in monolayer cultures as a challenge. No page numbers are available in our copy. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Xenotransplanted neurons reach much more advanced stages of spine maturation than reported so far in in vitro models of human corticogenesis, using adherent cultures or organoids.

    “The xenotransplanted neurons also reach much more advanced stages of spine maturation than reported so far in in vitro models of human corticogenesis, using adherent cultures or organoids (Astick and Vanderhaeghen, 2018, Di Lullo and Kriegstein, 2017, Lancaster and Knoblich, 2014)”

    From Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits (2019) · cites this paper as Di Lullo and Kriegstein, 2017

    The Challenges section describes brain organoids as relatively immature, and Establishing functional cortical circuits says circuit maturation remains a challenge. Neither passage compares dendritic-spine maturation in xenotransplanted neurons with adherent cultures or organoids, so the specific comparison is not supported. (LLM, conf=0.72)

    Experimental — accuracy not yet measured

  • APOE3Ch organoids stained prominently with Reelin, a marker of more mature neurons.

    “Consistent with a more mature phenotype, APOE3Ch organoids stained prominently with Reelin, a marker of more mature neurons (Figures 4A–D; Lancaster et al., 2013; Di Lullo and Kriegstein, 2017).”

    From APOE3 Christchurch modulates β-catenin/Wnt signaling in iPS cell-derived cerebral organoids from Alzheimer’s cases (2024) · cites this paper as Di Lullo and Kriegstein, 2017

    Experimental — accuracy not yet measured

  • Human brain organoids are a scaled-down, three-dimensional model of the human brain, mimicking various developmental features, including generation of new neurons by their conversion from radial glia and incorporation into neuronal network.

    “Human brain organoids are emerging as a scaled-down, three-dimensional model of the human brain, mimicking various developmental features, including generation of new neurons by their conversion from radial glia20 and incorporation into neuronal network21.”

    From Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB (2021) · cites this paper as [20]

  • Neural organoids present special challenges including a lack of reproducibility, batch-to-batch variation in transcriptional profiles, and susceptibility to small microenvironmental changes that may have a considerable effect on organoid fate.

    “Neural organoids present special challenges including a lack of reproducibility, batch-to-batch variation in transcriptional profiles, and susceptibility to small microenvironmental changes that may have a considerable effect on organoid fate165.”

    From Towards organoid culture without Matrigel (2021) · cites this paper as [165]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Study 2 is described as both externally patterned and signal-free

    Minor

    The paper calls the organoid model in reference 2 "extrinsic," meaning its patterning is influenced by externally added molecules. Earlier, it identifies reference 2 as a protocol that uses medium without neural-induction signalling molecules.

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Authors
Di Lullo, E., & Kriegstein, A. R.
Affiliations
University of California, San Francisco (US)
Corresponding author
Kriegstein, Arnold R.
ORCID
Kriegstein, Arnold R.
Venue
Nature Reviews Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2017-09-07
Volume
18
Issue
10
Pages
573-584
ISSN
  • 1471-003X (ISSN-L)
  • 1471-0048
DOI
10.1038/nrn.2017.107
PMC
PMC5667942
OpenAlex
W2753168648
Cited by
788
References
94
Citations by year
2026: 60 · 2025: 95 · 2024: 122 · 2023: 84 · 2022: 100 · 2021: 100 · 2020: 101 · 2019: 70 · 2018: 53 · 2017: 2
FWCI
5.79
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature reviews. Neuroscience (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institutes of Health
  • National Institute of Neurological Disorders and Stroke (R35NS097305)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
Keywords
brain organoids, neural development, human brain development, pluripotent stem cells, organoid formation, brain disease model, in vitro brain models
MeSH terms
Animals, Brain, Brain Diseases, Humans, Models, Neurological, Organoids, Pluripotent Stem Cells, Neurogenesis

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-01.

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