The use of brain organoids to investigate neural development and disease

In the paper

“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.”

What the sources say

• Partly supported

  The section on large-scale single-cell sequencing reports variation in cell-type production among organoids and a minority cluster expressing mesodermal markers. The Discussion also notes organoid-to-organoid variability. These passages support both observed outcomes, but attribute variability partly to bioreactor conditions; they do not establish that regional diversity causes either outcome. (LLM, conf=0.82)

  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. sourceExperimental — accuracy not yet measured
• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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
• Partly supported

  The Discussion (Comparison of Germinal Zones in Mouse and Monkey; page unavailable) reports a massively expanded outer subventricular zone in monkey and notes that radial-glial nuclei have been observed there. It speculates that radial glial cells are allocated to this zone, but does not establish that oRGs themselves are present in large numbers. The separate claim about human cortical evolution carries different citations in the citing sentence and is not attributed to this source. (LLM, conf=0.82)

  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). sourceExperimental — accuracy not yet measured
• Not checked yetcites [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
• Not checked yetcites [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
• Not checked yetcites [43] Dehay, C. & Kennedy, H. Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450 (2007). source
• Supported

  The Discussion (‘tRG cells are a common cell type in mammalian neocortex’; page unavailable) identifies outer radial glial (oRG) cells as translocating radial glial cells. The Results (‘The distribution of Pax6+ mitoses shifts to the oSVZ’; page unavailable) report that most Pax6+ mitotic precursors in developing macaque cortex were in the outer SVZ by E100, supporting the first clause. The evolutionary claim has a separate set of citations and is not attributed to this source in the citing sentence. (LLM, conf=0.88)

  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
• Supported

  The Conclusions identify oRG cells as self-renewing contributors to increased neuron number during the evolution of the human neocortex. The Diversity of Neocortical Foldedness section says oRG expansion in gyrencephalic species has been used to generate bigger, more complex brains. (LLM, conf=0.90)

  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 yetcites [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

In the paper

“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.”

What the sources say

• Supported

  The Results describe organoids derived from four families with idiopathic ASD and report increased FOXG1 expression and an increased proportion of GABAergic neurons. In “Altered neural proliferation and differentiation in organoids from ASD patients” (page unavailable), the authors report a shorter cell cycle in ASD-derived iPSCs and a similar trend in early neuronal progenitors; they did not find increased proliferation in more mature organoids. Thus, the cell-cycle finding applies to early developmental stages, rather than mature organoids. (LLM, conf=0.88)

  cites [83] Mariani, J. et al. FOXG1-Dependent dysregulation of GABA/Glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [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 yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [52] Luo, C. et al. Cerebral organoids recapitulate epigenomic signatures of the human fetal brain. Cell Rep. 17, 3369–3384 (2016). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“For example, a large number of cells were assigned to a mesodermal lineage, emphasizing the variability inherent in the protocol.”

What the sources say

• Not checked yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [31] Harrison-Uy, S. J. & Pleasure, S. J. Wnt signaling and forebrain development. Cold Spring Harb. Perspect. Biol. 4, a008094 (2012).

In the paper

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

In the paper

“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.”

In the paper

“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).”

In the paper

“Cerebral organoids grown from these cells contained fewer proliferating progenitor cells and showed premature neural differentiation compared to wild-type counterparts.”

What the sources say

• Not checked yetcites [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

In the paper

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

In the paper

“The brain organoid model is in an early phase of development.”

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

“another study reporting that AXL is a crucial receptor for infection of human glial cells (astrocytes and microglia) but not of human NPCs75.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

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

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

In the paper

“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”

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

“The amyloid hypothesis of AD posits that excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau84.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

In the paper

“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”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [78] Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221–225 (2011). source

In the paper

“The organoids also displayed severe apoptosis of neuroepithelial stem cells in the ventricular-like zone and a mitotic defect in oRGs50.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“However, they can also be used to discover human-specific phenotypes, providing an advantage over existing mouse models.”