Seminars in and xxx (xxxx) xxx–xxx

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Seminars in Cell & Developmental Biology

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Review Modeling neurological disorders using

Daniel Y. Zhanga, Hongjun Songb,c,d,e, Guo-li Mingb,c,d,f,* a Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA b Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA c Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA d Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA e The Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA f Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

ARTICLE INFO ABSTRACT

Keywords: Neurological disorders are challenging to study given the complexity and species-specific features of the Brain system. Brain organoids are three dimensional structured aggregates of neural that are generated by self- organization and differentiation from pluripotent stem cells under optimized culture conditions. These brain Neurological disease organoids exhibit similar features of structural organization and cell type diversity as the developing human Disease modeling brain, creating opportunities to recapitulate disease phenotypes that are not otherwise accessible. Here we re- 3D culture view the initial attempt in the field to apply brain organoid models for the study of many different types of human neurological disorders across a wide range of etiologies and pathophysiologies. Forthcoming advance- ments in both brain organoid technology as well as analytical methods have significant potentials to advance the understanding of neurological disorders and to uncover opportunities for meaningful therapeutic intervention.

1. Introduction With optimized culture conditions as well as a degree of intrinsic self- organization, brain organoids are able to capture certain aspects of the The human is one of the most complex tissues in tissue architecture and cell type diversity of the human nervous system biology, particularly in terms of its cell type diversity, cellular archi- through various stages of development. tecture and organization, and functional connectivity. Diseases related These features of brain organoids offer unique opportunities to to its development, degeneration, , and exposure to environ- model developmental processes and disease phenotypes not otherwise mental insults have been difficult to study due to the complexity of their available with alternative approaches, such as 2D monolayer and unique pathophysiologies and difficulties in accessing human speci- neurosphere cultures [8]. Certainly, brain organoids do not fully or mens. Animal models and human post-mortem analyses have been perfectly recreate the developing , and they also suffer absolutely invaluable; however, many aspects of brain development from a number of technical limitations; however, existing methods have and diseases exhibit essential human-specific features, and prospective proven adequate to recapitulate important features of diseases and studies remain nonetheless indispensable for mechanistic investigation yield valuable insights, and forthcoming advancements, such as in- and targeted interventions based on mechanistic understandings [1–3]. corporation of other non-neural cell types and vasculature, are expected Rapid progress in stem cell technologies over the past decades, in- to greatly improve the fidelity and scope of this model system. In this cluding reprogramming of somatic cells into induced pluripotent stem review, we focus on the use of brain organoids in modeling neurological cells (iPSCs) and differentiation of pluripotent stem cells (PSCs), in- diseases, highlighting the specific advantages of this model system as cluding both iPSCs and embryonic stem cells (ESCs), has widely en- well as discussing areas for further development. Overall, brain orga- abled access to human neural cells within an in vitro system that permits noids are a young and nascent field with demonstrated and substantial extensive experimental manipulation for phenotypic and mechanistic promise to deepen the understanding of basic neurobiology, to uncover studies [4,5]. These efforts have recently culminated in the develop- specific and actionable insights into neurological diseases, and to ad- ment of brain organoids, which are 3D structures that resemble the vance the development of effective therapeutics. developing nervous system and can be used as experimental models for studying normal and disease pathophysiology [6,7].

⁎ Corresponding author at: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. E-mail address: [email protected] (G.-l. Ming). https://doi.org/10.1016/j.semcdb.2020.05.026 Received 26 March 2020; Received in revised form 8 May 2020; Accepted 27 May 2020 1084-9521/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Daniel Y. Zhang, Hongjun Song and Guo-li Ming, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2020.05.026 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx

Table 1 Organoid models for congenital diseases with genetic etiologies.

Disease class Disease Mutation Patient derived/ genetically engineered References

Structural phenotypes ASPM (MCPH1) Patient-derived [21] WRD62 (MCPH2) Genetically engineered [20] CDK5RAP2 (MCPH3) Patient-derived [10] CPAP (Seckel syndrome) Patient-derived [22] CHMP1A (Microcephaly with pontocerebellar hypoplasia) Genetically engineered [23] TREX1 (Aicardi-Goutières syndrome) Patient-derived & Genetically engineered [24] Lissencephaly 17p13.3 deletion (Miller-Dieker syndrome) Patient-derived [27,28] LIS1 Genetically engineered [31] KATNB1 Genetically engineered [25] TUBA1A Genetically engineered [26] Periventricular heterotopia PLEKHG6 Genetically engineered [32] DCHS1 Patient-derived & Genetically engineered [33] FAT4 Neuropsychiatric disorders Schizophrenia DISC1 Genetically engineered [40] Patient-derived [18,42] 16p13.11 microduplication Patient-derived [44] FGFR1 Patient-derived [45] spectrum disorder Idiopathic Patient-derived [47] CHD8 Genetically engineered [48] CACNA1C (Timothy syndrome) Patient-derived [49] Intellectual disability Trisomy 21 (Down syndrome) Patient-derived [57] MECP2 (Rett syndrome) Patient-derived [58] Other Gangliosidosis GLB1 (GM1 gangliosidosis) Genetically engineered [59] HEXB (Sandhoff disease / GM2 gangliosidosis) Patient-derived [60] Pelizaeus-Merzbacher disease PLP1 Patient-derived [61]

2. Organoid models of early brain development Furthermore, some of the human-specific features of organoids can be captured in the presence and abundance of certain cell types, such as In less than a decade, brain organoids have emerged as novel and outer radial (oRGs) [11], which are relatively few in rodents [17]. useful tools for modeling normal human neural development and Finally, brain organoids are uniquely defined by their three-dimen- neurological disorders in a 3D in vitro culture system. Brain organoids sional architecture, often containing defined laminar zones of pro- are generated from PSCs, and with recent advancements in stem cell genitor cells and they give rise to [18]. A number of disorders technologies, species-specific – often human – as well as patient-specific involve defects in cellular organization and tissue architecture, and models of brain development and diseases are readily accessible. The brain organoids are well suited to model the underlying biological study of species differences and evolution lies outside the scope of this processes of these disorders. In all, the innate complexity of brain or- review and we focus on research efforts that use brain organoids from ganoids offers opportunities to study cellular processes and molecular patient-derived iPSCs or genome-edited human PSCs to study disease pathways that are essential to human diseases and yet not readily processes with the appropriate genetic backgrounds. available in alternative in vitro model systems. Protocols to generate brain organoids can be broadly divided into unguided and guided methods [6]. Unguided methods forgo the use of 3. Organoid models for congenital developmental disorders with targeted patterning factors and rely on intrinsic cues and self-organi- genetic etiologies zation capabilities of PSC aggregates under optimized growth condi- tions. Although this method generates an enormous diversity of cell 3.1. Structural abnormalities types, encompassing many areas of the brain, as well as , choroid plexus, and , the stochasticity and PSC line-to-line and or- One of the major advantages of brain organoids over existing 2D ganoid-to-organoid variability remain major challenges with this ap- culture models as well as neurospheres is the presence of cellular or- proach [9,10]. Guided methods target key signaling and morphogen ganization and cytoarchitecture that are formed through self-organi- pathways using small molecules and growth factors through at least the zation and the application of certain extracellular matrix factors. fi progenitor patterning stage to yield brain region-speci c organoids. Cerebral and forebrain organoids consist of neural rosette-like struc- Protocols to model many brain regions, including the forebrain, mid- tures with a fluid-filled ventricle at the center surrounded by sequential brain, hypothalamus, , and many others, have been re- layers of neural progenitor cells (NPCs) and neurons. These NPCs – ported [11 13]. As the brain organoid toolkit expands to encompass consist of various radial glia subtypes, and they demonstrate apical- more individual and combinations of brain regions, selection of an basal polarity, display typified mitotic translocation and cleavage an- appropriate model system will be important to best model the disorder gles, and act as a scaffold for neural migration. Thus, cortical organoids or biological process of interest. are uniquely useful for modeling diseases where structural defects are With both unguided and guided methods, brain organoids consist of prominent features of the clinical phenotype (Table 1). Future ad- ff a variety of cell types, including a number of di erent stem and pro- vancements may further elaborate the structural characteristics of fl genitor cells, neurons, and glia, all of which arise in sequence, re ecting cortical organoids especially those that emerge in later stages of de- the temporal dynamics of the genesis of these cells during normal brain velopment, such as the six neuronal layers of the neocortex and pro- development [11,14,15]. Thus, the emergence and diversity of these minent gyri and sulci along the cortical surface. cell types in brain organoids offer the opportunity to model phenotypes, such as proliferation and premature differentiation, as well as to ex- amine the cell type-specificeffects of various genetic mutations or en- 3.2. Microcephaly vironmental exposures. Brain organoid exhibit similar transcriptome, fi epitranscriptome and epigenome as the developing brain [11,14–16]. Microcephaly is clinically de ned as an occipitofrontal head cir- cumference of more than 2–3 standard deviations below the mean for

2 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx age and gender. Although the etiologies are diverse, most cases of patients with PH revealed a loss-of-function mutation in PLEKHG6, prenatal onset microcephaly are thought to involve impaired NPC specifically transcript variant 4 (PLEKHG6_4), which is associated with function and diminished overall neurogenesis. Brain organoids are oRGs and does not have an ortholog in mice [32]. Genetic manipulation important in modeling this disease process as mice lack the primate- of PLEKHG6_4 expression in cerebral organoids by led enriched and highly neurogenic outer subventricular zone (oSVZ) to altered neuronal migration, periventricular accumulation of neurons, where oRGs largely reside. This is thought to play a role in the more and disruption of apical surface integrity that influenced the behavior modest phenotypes observed in mouse models that bear genetic al- of adjacent un-manipulated neurons in a non-cell-autonomous manner. terations [19,20], whereas prominent phenotypes have been con- Mutations in protocadherins DCHS1 and FAT4, often in one allele, are sistently observed in models with causative patho- associated with PH, and cerebral organoids bearing these alterations genic mutations in a number of different genes, such as a centrosomal demonstrate the stereotypical feature of ectopic neuronal clusters near protein, CDK5RAP2, and a mitotic spindle protein, ASPM [10,21]. the ventricle [33]. In contrast, mice, even with homozygous knockout Studies in brain organoids have also identified a number of novel of Dchs1 and Fat4, do not demonstrate abnormal cortical development mechanisms and pointed towards potential therapeutic strategies. [34]. This is similar in lissencephaly, a disease also with neuronal mi- Centrosome dysfunction and disassembly impairment were gration defects, where genetic mutations found in patients can yield found in a cortical organoid model of Seckel syndrome, which is caused modest or no relevant phenotypes in mouse models [35]. Thus, brain by mutations in a centromeric protein, CPAP, also known as CENPJ organoid models offer the opportunity for investigating underlying [22]. Cilium disassembly impairment and NPC depletion were also mechanisms of neuronal migration, especially those that may not be found in cerebral organoids with mutations in centrosomal protein adequately recapitulated in other model systems. WRD62 [20]. Mutations in CHMP1A can lead to microcephaly with pontocerebellar hypoplasia, and cerebral organoid models demon- 3.5. Neuropsychiatric disorders strated that impaired Shh signaling can affect the maintenance of NPCs and pace of differentiation [23]. Aicardi-Goutières syndrome (AGS) can A number of neuropsychiatric disorders, including schizophrenia, arise from mutations in TREX1 and is an inflammatory disorder with autism spectrum disorder (ASD), and intellectual disability (ID) are severe neurologic deficits and microcephaly. Cerebral organoid models thought to involve important pathophysiological processes during early of AGS demonstrated smaller sizes and increased apoptosis in neuronal brain development [36]. Although the clinical focus in these diseases is regions, which were rescued upon treatment with reverse transcriptase on the behavioral outcomes, brain organoids may serve as useful inhibitors, Lamivudine and Stavudine [24]. models to study the molecular and cellular alterations that occur in early developmental stages. These diseases have complex and poorly 3.3. Lissencephaly understood genetic and environmental etiologies, and cases with cau- sative genetic alterations – though rare – have served as tractable Lissencephaly is characterized by the lack of gyri and sulci on the starting points to build informative disease models. and is thought to be due to impaired neuronal migra- tion. Although animal studies with genetically manipulated mice have 3.6. Schizophrenia been informative, their intrinsic lissencephaly places clear limitations on the fidelity of these models, and iPSC-derived brain organoids may Schizophrenia is a severe mental disorder affecting numerous do- bridge the gap to the human disease. Neuronal migration defects were mains of psychological function, and although its onset is in early found in cerebral organoids with pathogenic mutations in microtubule adulthood, a number of neurodevelopmental events remain closely proteins KATNB1 and TUBA1A as well as a cerebral organoid model of linked with its pathogenesis. DISC1 is a scaffolding protein that inter- Miller-Dieker syndrome (MDS) [25–27]. This MDS model also revealed acts with many different pathways and its rare mutations have been a reduced NPC pool due to increased apoptosis and reduced vertical genetically linked to a number of major psychiatric disorders, including divisions, oRG-specific mitotic defects, and imbalanced NPC prolifera- schizophrenia [37]. Prior studies in mouse, zebrafish, and other cell tion and differentiation [28]. culture models have demonstrated that DISC1 mutations lead to dys- The gyri and sulci in the human brain are largely formed in the third regulated WNT signaling and neural progenitor activity [38,39]. Brain trimester; however, it remains difficult to model this later period in organoids modeling a chromosomal translocation disrupting the DISC1 prenatal development using brain organoids [29]. Most brain organoids locus demonstrated morphological changes and reduced proliferation, do not bear prominent gyrification, which may be due to a number of which could be rescued with WNT pathway antagonism and phe- different challenges, such as inadequate organoid maturation; however, nocopied by WNT pathway agonism [40]. Another DISC1 brain orga- novel approaches have been pursued to capture aspects of this essential noid model with a rare 4 base-pair frame-shift mutation found in major feature of the human brain. PTEN mutation leads to NPC expansion and psychiatric disorder patients [41] revealed that disruption of the surface folding in brain organoids, which was only observed with DISC1/NDEL1 complex leads to delayed cell-cycle progression and re- human, but not mouse, cell lines [30]. Although non-physiologic, this duced proliferation, consistent with studies in mouse models and 2D mutant background may be useful in certain circumstances to study cell culture models [42]. A sliced forebrain cortical organoid model was gyri fication and model lissencephalies. Alternatively, a brain-organoid- recently developed, which exhibits distinct upper and deep cortical on-a-chip approach was used to study the biomechanics of folding and layers with segregation of neuronal subtype markers [18]. In this identified contraction at the organoid core and expansion at the peri- model, the frameshift DISC1 mutation leads to deficits in cortical meter as opposing forces leading to wrinkling [31]. Brain organoid subtype differentiation and failure of segregation of upper and models with alterations in LIS1, which is located within the chromo- deep cortical neuronal layers [18]. Notably, dysregulation of the la- some 17 deletion locus in MDS and is strongly associated with lissen- minar expression of neuronal subtype marker genes has been observed cephaly, demonstrated fewer wrinkles than isogenic controls and al- in from children with ASD [43]. Cerebral organoids with chro- tered biomechanical properties. mosome 16p13.11 microduplication demonstrated reduced prolifera- tion as well as dysregulation of the NFκB p65 pathway, which could be 3.4. Periventricular heterotopia restored using pharmacologic agents and genetic manipulations to rescue the proliferation phenotype [44]. Cerebral organoids from pa- Periventricular heterotopia (PH) is a condition where impaired tients with schizophrenia but without any canonical alterations de- neuronal migration leads to heterotopic neurons residing adjacent to monstrated aberrant cortical organization, which could be linked to the ventricles. Examination of de novo coding variants identified in nuclear FGFR1 signaling and phenocopied with pharmacologic

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Table 2 Organoid models for congenital diseases with environmental exposures.

Disease class Exposure References

Infection Zika virus [11,67,68,70,71,72,73,74,75,76,77,78,79,80,81,82,83,148] Japanese encephalitis virus [84] Cytomegalovirus [86,87] Chemical agents & radiation Hypoxia [89,90,91] Alcohol [92] Cocaine [93] Nicotine [94] 5-MeO-DMT [95] Tranylcypromine [102] Imadazole antifungals [103] Bisphenol A [11] Gamma irradiation [104] inhibition of this pathway [45]. Overall, these models of schizophrenia syndrome demonstrated impaired neuronal differentiation, which could have yielded insights into the cellular processes associated with disease- be recapitulated with MECP2 knockdown and were linked to increased associated variants, especially into defects in cellular organization and levels of miR-199 and miR-214, two miRNAs that impair neuronal morphogenesis that would not be well captured in other cell culture differentiation by altering ERK and AKT signaling [58]. models. However, large domains of analyses, such as alterations in neuronal network connectivity, remain largely unexplored. 3.9. Other diseases

3.7. Autism spectrum disorder 3.9.1. Gangliosidosis Gangliosidosis is a type of lysosomal storage disorder where gang- ASD is a neurodevelopmental disorder characterized by defects in liosides accumulate aberrantly due to inborn errors of metabolism. social communication and social behaviors. Multiple lines of evidence Organoid models of GM1 and GM2 gangliosidosis demonstrate accu- in mouse models and human patients have implicated interneurons in mulation of these lipids, which can lead to cerebral organoid degen- the pathogenesis of this disease [46], and brain organoid models from eration and be rescued with targeted gene therapy [59,60]. different genetic backgrounds have been consistent with this prior knowledge and expanded upon them. For example, brain organoid 3.9.2. Pelizaeus-Merzbacher disease models of idiopathic ASD exhibited increased production of GABAergic Pelizaeus-Merzbacher disease (PMD) is an X-liked monogenic leu- neurons, which was in part attributable to increased expression of kodystrophy caused by a mutation in PLP1, leading to defects in myelin FOXG1 [47]. This study highlights an advantage of brain organoids as production. Oligocortical spheroids were developed by supplementa- mouse and other cell culture models would not be suited to examine tion with oligodendrocyte lineage promoting factors, and models of phenotypes involving a number of different cell types within an un- PMD showed phenotypes of perinuclear accumulation of PLP1 and re- determined causal genetic background. Cerebral organoids with muta- duced oligodendrocytes, which could be rescued with PERK inhibitors tions in CHD8, a chromatin remodeler and a ASD risk gene, showed [61]. dysregulated expression of genes important in GABAergic neuron dif- ferentiation, consistent with results from the study of idiopathic cases 3.10. Organoid models for congenital diseases with environmental [48]. A novel model of Timothy syndrome, a disease that shares so- exposures cialization and communication phenotypes with ASD, bearing muta- tions in CACNA1C was developed by fusing cortical and subpallium 3.10.1. Viral infection spheroids into an assembloid, which then demonstrated impaired in- A number of infectious diseases have prominent neural and neuro- terneuron saltation and mobility that could be rescued upon treatment development sequelae that can lead to lasting deficits, and brain or- with L-type calcium channel inhibitors [49]. Future studies examining ganoids have played an important role in studying these diseases, the network integration of these interneurons and resulting excitation- particularly in Zika virus infection (Table 2). One area of particular inhibition balance may build on this body of work. advantage is the cellular diversity within brain organoids, which offers unique opportunities to examine viral tropism as well as to study cell 3.8. Intellectual disability type-specific responses to these exposures. As brain organoids lack immune cells, and therefore are not suitable for understanding pa- Intellectual disability is a neurodevelopmental disorder with deficits thogen-host immune responses. Mouse models of viral infections are across a broad spectrum of intellectual function. Down Syndrome (DS) advantageous in this aspect, but lacking species specificity of essential is caused by amplification of chromosome 21 leading to developmental viral functions [62,63]. and intellectual disabilities. OLIG1 and OLIG2 are located on chromo- some 21 and have been previously implicated in DS [50–52]; however, 3.10.2. Zika virus the expression patterns of these two transcription factors differ between Zika virus is a flavivirus that is primarily transmitted by mosquitos, mouse and human, where in humans OLIG2, but not OLIG1, is abun- and although the symptoms of infection in adults are mild, prenatal dantly expressed in populations of neural progenitors and is thought to exposure and infection have been linked to severe neurodevelopmental play a more important role in DS [53–56]. Brain organoid models of DS defects, including microcephaly. Since the 2015 outbreak of Zika Virus showed overproduction of OLIG2-expressing ventral forebrain pro- in Central and South America, brain organoids have been used by nu- genitors and GABAergic interneurons [57]. Injection of dissociated or- merous research groups as a leading model system to identify cellular ganoid cells into neonatal mice led to increased GABAergic neurons and tropism, mechanisms of infection, and potential therapeutic options behavioral deficits, both of which could be reversed with knockdown of [64]. This body of research highlights how leveraging the unique fea- OLIG2, thus better defining the link between OLIG1/2, interneuron tures of brain organoids, such as its cell type diversity and structural development, and DS related phenotypes. Organoid models of Rett organization, can lead to important insights in domains ranging from

4 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx basic disease biology to drug discovery [65,66]. 3.11.1. Hypoxia Early studies using brain organoid models of Zika virus infection Prenatal hypoxic injury is a common cause of neurodevelopmental were the first to reveal viral tropism for NPCs, leading to reduced deficits as even transient episodes can cause cellular damage that lead overall size of organoids and numbers of NPCs and neurons [11,67,68]. to long term neuropsychiatric sequelae. Prior studies in mouse models AXL was proposed as a candidate viral entry receptor given its enriched identified SOX2-expressing cells at the basal SVZ that form a proposed expression in radial glia cells, however, subsequent studies in AXL oSVZ-like cell layer and are sensitive to oxygen levels during develop- knockout brain organoids showed largely unchanged susceptibility to ment [88]. However, given the lack of a true oSVZ in mice and species- Zika virus infection, suggesting that AXL is not essential for viral entry related differences in cortical progenitor pools, the implications of these [69,70]. Co-culture models of iPSC-derived microglia-like cells with findings to human pathophysiology remain unclear. Brain organoid neural spheroids and human primary monocytes with brain organoids models of hypoxia show reduced numbers of progenitor cells, including demonstrated that these non-neural cells may be suitable viral carriers oRGs and intermediate progenitors, as well as substantial tran- and mediate dissemination into the developing brain [71,72]. Zika scriptomic alterations, which can be rescued upon reoxygenation, virus infection of NPCs leads to reduced proliferation and premature treatment with an integrated stress response inhibitor, ISRIB, or treat- differentiation, and is also characterized by altered division planes, ment with minocycline [89–91]. Endothelial cells and other elements of impaired centrosome function, disrupted apical adherens junctions, cerebrovascular biology play an essential role in hypoxic injury, and disorganized radial glia morphology, and activation of TLR3 signaling brain organoids that incorporate these components may offer a more pathways [73–75]. NS2A protein encoded by the Zika virus genome complete model system in studying this disease process in the future. was found to disrupt adhereins junctions of radial glia in forebrain organoids, resulting in reduced proliferation and premature differ- 3.11.2. Drugs of abuse: alcohol, cocaine, nicotine, and 5-MeO-DMT entiation [76]. Furthermore, Zika virus infected brain organoids Prenatal exposure to drugs of abuse is widely prevalent and can lead showed differential DNA methylation at loci associated with neurode- to long lasting changes in brain development. Exposure of brain orga- velopmental and psychiatric disorders, suggesting the possibility of noids to ethanol, cocaine, and nicotine yielded common phenotypes of long-term consequences [77]. Finally, a number of drug screens fol- premature neuronal differentiation and abnormal neurite outgrowth lowed with validation in brain organoids have identified compounds [92–94]. Notably, exposure to cocaine, a stimulant that inhibits the such as emricasan, hippeastrine, 25-hydroxycholesterol and certain reuptake of monoamine neurotransmitters, also led to CYP3A5 induced antibiotics and antivirals as promising candidates for treating Zika virus production of reactive oxygen species. 5-MeO-DMT is a serotonin-like infection [78–83]. molecule with psychoactive and hallucinogenic properties, and ex- posure in brain organoids lead to dysregulation of proteins associated 3.10.3. Japanese encephalitis virus (JEV) with inflammation, long term potentiation, and cytoskeletal dynamics Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that [95]. Many of these drugs of abuse have neuromodulatory activity, and is the leading cause of viral encephalitis in Asia, and although symp- studying these effects on the different neuronal populations at different tomatic infection is uncommon, neurologic sequelae can be severe and degrees of maturation may yield insights to how early exposure lead to long lasting. Cortical organoids models of JEV revealed viral tropism for the observed clinical behavioral phenotypes. astrocytes and NPCs, especially oRGs, leading to reduced organoid size and increased cell death [84]. Interestingly, older organoids exhibited 3.11.3. Pharmacologic agents less susceptibility to JEV infection as well as greater activation of in- The large majority of pharmaceutical drugs have an unknown risk in terferon signaling pathways. pregnancy despite their necessity and widespread use in pregnant women [96]. Mouse models are enormously useful but also complicated 3.10.4. Cytomegalovirus (CMV) by host metabolism, maternal-fetal biology, and species differences in CMV is a ubiquitous pathogen, and prenatal infection can lead to key developmental programs [97,98]. The impact of these challenges is neurodevelopmental defects, including microcephaly; however a major exemplified by the antiemetic, thalidomide, which yields almost no challenge of studying CMV is its strict species specificity [85]. Brain teratogenicity in mice but severe deformities in humans [99, 100]. In organoid models of CMV infection demonstrated microcephaly, dis- cerebral organoid models, vincristine, a microtubule destabilizing anti- rupted cellular organization, and abnormal calcium signaling, which cancer agent, and tranylcypromine, a monoamine oxidase inhibitor, could be prevented with neutralizing antibodies or partially rescued both yielded reduced neural and glial cell numbers [101,102]. Further with maribavir, an experimental antiviral agent [86,87]. genomic analyses found that vincristine exposure dysregulates extra- Brain organoid modeling has led to significant novel insights into cellular matrix associated genes, whereas tranylcypromine exposure viral infection of the developing nervous system, especially with respect increases H3K4 dimethylation. In contrast, exposure to imidazole an- to cell type tropism, morphological defects, and potential novel thera- tifungals leads to the generation of oligodendrocytes and remyelination pies. Future incorporation of additional cell types, such as microglia through the inhibition of CYP51 and the subsequent accumulation of and lymphoid cells, into the brain organoid model in a biologically lanosterol [103]. Although these case studies of individual drugs de- faithful manner will help to better understand complex host-pathogen monstrate the potential of brain organoids, scaling to mid- or high- interactions and their consequences. throughput screens for teratogenicity will require high volume sample processing pipelines as well as suitable phenotypic standards for iden- 3.11. Chemical agents and radiation tifying toxicity.

The developing brain is often susceptible and sensitive to the ex- 3.11.4. Toxins and radiation posure or depletion of many different non-biological agents, including Toxin and radiation exposures are ubiquitous in the environment, oxygen, drugs of abuse, pharmacological agents, frank toxins, and ra- and brain organoids can be useful in understanding their toxicity, me- diation. As with infectious diseases, modeling these non-biological ex- chanisms, and neurologic sequelae. Gamma irradiation of cerebral or- posures in brain organoids presents an important advantage of studying ganoids leads to a thinned neuroepithelium and reduced metabolic differential susceptibly and responses in a system where all these dif- output that could be rescued by pretreatment with rapamycin or sub- ferent cell types are integrated in a biologically meaningful manner. sequent treatment with minocycline [104]. Bisphenol A is a component These published research studies lay the groundwork for future op- in many plastics and its exposure in forebrain organoids at high doses portunities to screen for developmental neuro-teratogenicity across leads to reduced neural proliferation and thinned ven- broad drug development and environmental applications (Table 2). tricular zone thickness [11]. Expanding this to larger scale, a screen of

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Table 3 Organoid models for neurodegenerative diseases.

Disease Mutation Patient derived/ genetically engineered References

Alzheimer's disease APP duplication Patient derived [114] PSN1 Patient derived Patient derived [116] Patient derived [115] Trisomy 21 Patient derived APOE4 Genetically engineered [117] Genetically engineered [118] Wild type with Aftin-5 treatment Patient derived [119] Frontotemporal dementia MAPT Patient derived [121] Huntington's disease HTT Patient derived [124] Creutzfeldt-Jakob disease Wild type with CJD brain homogenate exposure Patient derived [125] Hereditary spastic paraplegia SPG11 Patient-derived [126] compounds known to be toxic and non-toxic in brain organoids was disease, however, the lack of cellular components such as microglia and analyzed by RNA sequencing and used to generate a linear support vasculature, as well as the relatively low synaptic activity and cellular vector classifier that had a > 90 % classification accuracy [105]. immaturity are important challenges that will require ongoing evalua- tion and innovation [120]. 3.12. Organoid models for neurodegenerative diseases 3.12.2. Frontotemporal dementia Although brain organoids are more often designed and thought of as Frontotemporal dementia is a common type of early-onset neuro- models of brain development, their application towards studying neu- degenerative disease with behavioral, language, and motor impair- rodegeneration have yielded models that recapitulate certain essential ments. Mechanistic studies in brain organoids validated an important phenotypes, such as the presence of misfolded protein aggregates mechanistic association between p25, a disease-related fragment of p35 (Table 3). Notably, secreted proteins and extracellular deposits may that enhances CDK5 activity, and tau phosphorylation in a human accumulate to higher levels in the interstitial spaces in this model system that had been previously observed in mouse models [121–123]. system simply due to the 3D aggregation of these cells even without the typified structure of brain organoids [106]. With the growing appre- 3.12.3. Huntington’s disease ciation of the prion-like behavior observed in these diseases, studying Huntington’s disease (HD) is an inherited neurodegenerative disease neuronal diversity and connectivity may contribute to understanding caused by excessive CAG repeats in the huntingtin gene. Brain organoid how these pathologies may spread and disseminate. Neuronal and glial models demonstrated poorer neuroecoderm patterning with increasing maturation as well as the potential interference of progenitor cells are CAG expansion, disrupted cortical differentiation and organoid cy- concerns for the validity of brain organoids as models of neurodegen- toarchitecture, which could be partially rescued with knockdown of erative diseases, and they represent areas for further investigation and HTT and pharmacologic inhibition of ADAM10 [124]. development. 3.12.4. Creutzfeldt-Jakob disease 3.12.1. Alzheimer’s disease Creutzfeldt-Jakob disease (CJD) is a rare, fatal, and fast-progressing Alzheimer’s disease (AD) is an age-related neurodegenerative dis- neurodegenerative disease that is caused by abnormal forms of prion ease characterized by neuronal cell loss, amyloid aggregation, and hy- protein. Wild-type cerebral organoids were exposed to brain homo- perphosphorylated tau. Transgenic mouse models exist for AD that genates from two patients with sporadic CJD, leading to prion protein mimic a range of pathologies [107,108], however, a number of species- uptake and propagation [125]. specificdifferences, such as the expression of tau isoforms [109,110], and failed clinical trials for drugs that showed promising results in mice 3.12.5. Hereditary Spastic Paraplegia have prompted the development of human brain organoid models that Hereditary spastic paraplegia is characterized by progressive de- also have the added benefit of adaptability to high throughput drug generation of the corticospinal tracts. Cerebral organoid models with screening [111–113]. Brain organoid models of familial AD with mu- SPG11 mutations demonstrated premature differentiation, reduced tations in APP or PSN1 yield amyloid aggregates, hyperphosphorylated overall proliferation, and larger ventricles, which could be partially tau, endosome abnormalities, and increased inflammatory markers, rescued with GSK3β inhibitors [126]. which could in part be rescued with β- and γ-secretase inhibitors [114–116]. Models of sporadic AD showed that APOE4 brain organoids 4. Organoid models for cancer exhibit increased Aβ puncta and phosphorylated tau, as well as upre- gulation of neurogenic genes [117,118]. Beyond genetic models of AD, Organoid models of cancer have emerged across a number of dif- treatment of wild-type brain organoids with amyloidogenic Aftin-5 led ferent tumor types and organ systems to better preserve the cellular to increased levels of Aβ42 and Aβ oligomers [119]. These brain or- heterogeneity and microenvironment in an in vitro system (Table 4). ganoid models of AD capture essential biochemical features of the Some brain tumor organoid models are similar to brain organoids in

Table 4 Organoid models for cancer.

Disease Mutation Patient derived/ genetically engineered References

Tuberous Sclerosis TSC1 & TSC2 Genetically engineered [135] Glioma HRAS & TP53 Genetically engineered [136] MYC, CDKN2A, CDKN2B, EGFR, EGFRvIII Genetically engineered [137] Various Patient derived [138,139,140,141,142,144,145,146]

6 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx that they are generated from PSCs with defined genetic alterations, tumor, including histology, transcriptome, and genetic mutation land- however, other models are derived from primary surgical tissue and scapes. These organoids also show rapid and aggressive infiltration contain patient-specific genetic features. Notably, the patient-derived once transplanted into the brain of immunodeficient mice, and they organoids contain many different types of non-neoplastic cells, in- have been used to test personalized treatment responses to targeted cluding immune cells, creating opportunities to study these intercellular drug therapies and immunotherapy. Additional GBM organoid models interactions and how they might influence tumor growth or response to have been developed by introducing GBM cells into 3-D printed neural treatment. These emerging models offer unique opportunities to study organoids or iPSC-derived brain organoids [144–146]. By placing tumorigenesis, cellular heterogeneity, tumor cell migration, and re- tumor cells within a neuroanatomically appropriate human micro- sponse to therapies. environment, these hybrid organoids may better recapitulate the in- trinsic cellular states found in GBM [147]. The biology of GBM is 4.1. Genetically modified models heavily influenced by its microenvironment, and inclusion of these cellular components, either by preserving these cells from patient-de- 4.1.1. Benign tumors rived tissue or by introducing them exogenously, will help to generate a Tuberous sclerosis complex (TSC) is a genetic neurocutaneous dis- more complete model of this disease. order characterized by multiple benign hamartomas in various organ systems as well as severe neurological symptoms including seizures. 5. Prospective Mouse models of TSC have offered substantial insight into a number of disease phenotypes, such as epilepsy and altered neuronal differentia- Brain organoids are a model system still in early phases of devel- tion [127–129]; however, almost all lack the hallmark cortical tubers opment, establishment, and application. Many methodology-oriented [130–132], and those with focal lesions lack histopathologic findings, studies advocate for the inherent validity of brain organoids with a such as true giant cells and astrogliosis [133,134]. Cortical organoid combination of imaging, transcriptomic, and electrophysiological ana- models with mutations in TSC1 or TSC2 have dysregulated mTORC1 lyses. This multifaceted approach is largely convincing; however, or- signaling during neurogenesis, forming cortical tuber-like structures ganoid data are rarely, if ever, a perfect match with existing datasets of with large numbers of astrocytes, which can be attenuated with the developing human brain. This is hardly surprising for numerous mTORC1 inhibition [135]. reasons, such as that organoids are generated in vitro without many essential components, including a blood supply and endothelial cell 4.1.2. Malignant tumors capillary network as well as functional immune system and microglia. Tumorigenesis is thought to be initiated by a series of genetic events Complementing this first-principles approach, numerous studies leading to the gain of cancer hallmarks, such as uncontrolled pro- have shown evidence of brain organoid empirical validity across a wide liferation and ability to invade normal tissues. Genetically defined range of diseases. For example, brain organoids of congenital and in- models of cancer offer the opportunity to interrogate the relationship fectious microcephaly show phenotypes of reduced organoid size and between specific mutations and tumorigenesis, as well as a tumor model reduced progenitor proliferation, which are consistent with clinical with a defined and controlled genetic background. Introduction of a observations and known disease processes [11]. As another example, mutant HRas G12 V by homologous recombination into the gene body patient-derived brain tumor organoids show in vitro drug sensitivity of TP53 in cerebral organoids leads to generation of rapidly pro- that is consistent with their genetic features and in vivo responses [142]. liferative cells that invade and take over the entire organoid [136]. These kinds of empirical observations and clinical correlates contribute These cells demonstrate a mesenchymal gene expression signature and strongly to the evidence that these organoid models can recapitulate are tumorigenic and invasive upon orthotopic xenograft into im- certain important aspects of the disorder with reasonable fidelity, and munodeficient mice. Another model features MYC overexpression and thus they lend confidence to the application of brain organoids in – – – – CDKN2A / /CDKN2B / /EGFROE/EGFRvIIIOE to yield rapidly pro- modeling neurological disorders. liferative cells that could expand and invade upon xenograft into im- Brain organoids offer some unique opportunities over other existing munodeficient animals [137]. These organoids responded to targeted model systems, such as control over the genetic background, structural therapies in a mutation-dependent manner, validating that these mu- architecture, and intrinsic cell-type diversity. Stem cell technologies tations were indeed driving tumor-like behavior and that these orga- have allowed for access to human- and patient-specific samples, and noids could be used to study drug effects in the context of specific ongoing studies, especially those of mental disorders without a known genomic variants. genetic etiology, would benefit from models derived from idiopathic cases. Organoid structure is well characterized by imaging methods as 4.2. Patient-derived models there are established immunohistochemical markers delineating key cytoarchitectural landmarks, and advancements in 3D volumetric Organoids generated from patient-derived cells and tissue may imaging, such as those involving tissue clearing methods, may offer better recapitulate the genetic and cellular features of the originating additional insights. The cellular diversity in brain organoids represents parent tumor as compared with genetically engineered models. In one another unique advantage of this model system, and advances in multi- (GBM) organoid model, patient tissue is dissociated or modal and massively multiplexed single-cell technologies will allow for minced and then embedded into in the presence of EGF/bFGF better characterization of the cell type-specificeffects of different dis- to yield organoids that can be readily expanded and xenografted into ease processes. Electrophysiology and network connectivity are less immunodeficient mice [138]. These organoids responded to treatment commonly examined in brain organoids, and future studies may benefit with the MSI1 inhibitor, Luteolin, and Zika virus infection [139,140]. In from specialized tools and instrumentation suitable for these relatively this same model, quiescent H2B-GFP cells were identified after dox- small 3D structures. Overall, advancements in analytical methods ycycline induction and long term chase periods and found to exhibit a across a broad range of modalities will allow for better leveraging of the mesenchymal-like gene expression pattern that was characterized by unique advantages and attributes of brain organoids. Correspondingly, metabolic adaptions and extracellular matrix interactions as well as the broadening repertoire of brain organoids across different develop- signatures of hypoxia and TGFβ signaling [141]. A new GBM organoid mental stages and specific brain regions will also help in the study of model was recently developed by cutting tumor tissue into small pieces different neurological disorders within the best possible biological that were cultured in a chemically defined medium without addition of context. EGF/bFGF or extracellular matrix [142,143]. These organoids maintain In addition to these technical challenges that are currently being cellular heterogeneity and exhibit many features similar to the original addressed by this rapidly growing field, brain organoids face some

7 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx intrinsic limitations in modeling neurological disorders. Organoid X. Zhuang, S. Canzar, C. He, G.L. Ming, H. Song, Temporal control of mammalian models of neuropsychiatric disorders have so far been focused on the cortical neurogenesis by m(6)A methylation, Cell 171 (4) (2017) 877–889 e17. [17] J.H. Lui, D.V. Hansen, A.R. Kriegstein, Development and evolution of the human mechanistic connections between the underlying genetic alterations neocortex, Cell 146 (1) (2011) 18–36. and the observed cellular and molecular changes. However, some of the [18] X. Qian, Y. Su, C.D. Adam, A.U. Deutschmann, S.R. Pather, E.M. Goldberg, K. Su, major challenges are that these disorders can manifest in behavioral S. Li, L. Lu, F. Jacob, P.T.T. Nguyen, S. Huh, A. Hoke, S.E. Swinford-Jackson, Z. Wen, X. Gu, R.C. Pierce, H. Wu, L.A. Briand, H.I. Chen, J.A. Wolf, H. Song, phenotypes that are likely impossible to assess in any in vitro system, G.L. Ming, Sliced human cortical organoids for modeling distinct cortical layer and the broad clinical relevance of observed organoid phenotypes are formation, Cell Stem Cell (2020). unclear. Nonetheless, brain organoids already represent a major leap [19] J.N. Pulvers, J. Bryk, J.L. Fish, M. Wilsch-Bräuninger, Y. Arai, D. Schreier, forward in modeling neurological diseases, and forthcoming innova- R. Naumann, J. Helppi, B. Habermann, J. Vogt, R. Nitsch, A. Tóth, W. Enard, S. Pääbo, W.B. Huttner, Mutations in mouse < em > Aspm < /em > (abnormal tions as well as growing application of this model system to a broad spindle-like microcephaly associated) cause not only microcephaly but also major range of disease contexts will undoubtedly yield exciting new findings. defects in the germline, Proc. Natl. Acad. Sci. 107 (38) (2010) 16595–16600. [20] W. Zhang, S.L. Yang, M. Yang, S. Herrlinger, Q. Shao, J.L. Collar, E. Fierro, Y. Shi, A. Liu, H. Lu, B.E. Herring, M.L. Guo, S. Buch, Z. Zhao, J. Xu, Z. Lu, J.F. Chen, Declaration of Competing Interest Modeling microcephaly with cerebral organoids reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly in neural progenitors, Nat. Commun. 10 The authors declare no competing interests. (1) (2019) 2612. [21] R. Li, L. Sun, A. Fang, P. Li, Q. Wu, X. Wang, Recapitulating cortical development with organoid culture in vitro and modeling abnormal spindle-like (ASPM related Acknowledgment primary) microcephaly disease, Protein Cell 8 (11) (2017) 823–833. [22] E. Gabriel, A. Wason, A. Ramani, L.M. Gooi, P. Keller, A. Pozniakovsky, I. Poser, ’ F. Noack, N.S. Telugu, F. Calegari, T. Saric, J. Hescheler, A.A. Hyman, We thank K.M. Christian for comments. The research in the authors M. Gottardo, G. Callaini, F.S. Alkuraya, J. Gopalakrishnan, CPAP promotes timely laboratories were supported by grants from the National Institutes of cilium disassembly to maintain neural progenitor pool, EMBO J. 35 (8) (2016) Health (R37NS047344 and R35NS1166843 to H.S., R35NS097370 and 803–819. [23] M.E. Coulter, C.M. Dorobantu, G.A. Lodewijk, F. Delalande, S. Cianferani, U19AI131130 to G-l.M.), SFARI (to H.S.), and the Sheldon G. Adelson V.S. Ganesh, R.S. Smith, E.T. Lim, C.S. Xu, S. Pang, E.T. Wong, H.G.W. Lidov, Medical Research Foundation (to G-l.M.). D.Y.Z. was partially sup- M.L. Calicchio, E. Yang, D.M. Gonzalez, T.M. Schlaeger, G.H. Mochida, H. Hess, ported by the Blavatnik Family Fellowship in Biomedical Research. W.A. Lee, M.K. Lehtinen, T. Kirchhausen, D. Haussler, F.M.J. Jacobs, R. Gaudin, C.A. Walsh, The ESCRT-III protein CHMP1A mediates secretion of sonic hedgehog on a distinctive subtype of extracellular vesicles, Cell Rep. 24 (4) (2018) References 973–986 e8. [24] C.A. Thomas, L. Tejwani, C.A. Trujillo, P.D. Negraes, R.H. Herai, P. Mesci, A. Macia, Y.J. Crow, A.R. Muotri, Modeling of TREX1-dependent autoimmune [1] M. Hafezparast, A. Ahmad-Annuar, N.W. Wood, S.J. Tabrizi, E.M. Fisher, Mouse disease using human stem cells highlights L1 accumulation as a source of neu- models for neurological disease, Lancet Neurol. 1 (4) (2002) 215–224. roinflammation, Cell Stem Cell 21 (3) (2017) 319–331 e8. [2] T. Kaiser, G. Feng, Modeling psychiatric disorders for developing effective treat- [25] M. Jin, O. Pomp, T. Shinoda, S. Toba, T. Torisawa, K. Furuta, K. Oiwa, ments, Nat. Med. 21 (9) (2015) 979–988. T. Yasunaga, D. Kitagawa, S. Matsumura, T. Miyata, T.T. Tan, B. Reversade, [3] R.E. McCullumsmith, J.H. Hammond, D. Shan, J.H. Meador-Woodruff, S. Hirotsune, Katanin p80, NuMA and cytoplasmic cooperate to control Postmortem brain: an underutilized substrate for studying severe mental illness, microtubule dynamics, Sci. Rep. 7 (2017) 39902. Neuropsychopharmacology 39 (1) (2014) 65–87. [26] Y. Bamba, Y. Kanemura, H. Okano, M. Yamasaki, Visualization of migration of [4] Y. Shi, H. Inoue, J.C. Wu, S. Yamanaka, Induced pluripotent stem cell technology: human cortical neurons generated from induced pluripotent stem cells, J. a decade of progress, Nat. Rev. Drug Discov. 16 (2) (2017) 115–130. Neurosci. Methods 289 (2017) 57–63. [5] Z. Wen, K.M. Christian, H. Song, G.L. Ming, Modeling psychiatric disorders with [27] M. Bershteyn, T.J. Nowakowski, A.A. Pollen, E. Di Lullo, A. Nene, A. Wynshaw- patient-derived iPSCs, Curr. Opin. Neurobiol. 36 (2016) 118–127. Boris, A.R. Kriegstein, Human iPSC-derived cerebral organoids model cellular [6] X. Qian, H. Song, G.L. Ming, Brain organoids: advances, applications and chal- features of lissencephaly and reveal prolonged mitosis of outer radial glia, Cell lenges, Development 146 (8) (2019). Stem Cell 20 (4) (2017) 435–449 e4. [7] E. Di Lullo, A.R. Kriegstein, The use of brain organoids to investigate neural de- [28] V. Iefremova, G. Manikakis, O. Krefft, A. Jabali, K. Weynans, R. Wilkens, velopment and disease, Nat. Rev. Neurosci. 18 (10) (2017) 573–584. F. Marsoner, B. Brandl, F.J. Muller, P. Koch, J. Ladewig, An organoid-based model [8] J.W. Adams, F.R. Cugola, A.R. Muotri, Brain organoids as tools for modeling of cortical development identifies Non-cell-Autonomous defects in wnt signaling human neurodevelopmental disorders, Physiology (Bethesda) 34 (5) (2019) contributing to miller-dieker syndrome, Cell Rep. 19 (1) (2017) 50–59. 365–375. [29] T. White, S. Su, M. Schmidt, C.Y. Kao, G. Sapiro, The development of gyrification [9] G. Quadrato, T. Nguyen, E.Z. Macosko, J.L. Sherwood, S. Min Yang, D.R. Berger, in childhood and adolescence, Brain Cogn. 72 (1) (2010) 36–45. N. Maria, J. Scholvin, M. Goldman, J.P. Kinney, E.S. Boyden, J.W. Lichtman, [30] Y. Li, J. Muffat, A. Omer, I. Bosch, M.A. Lancaster, M. Sur, L. Gehrke, Z.M. Williams, S.A. McCarroll, P. Arlotta, Cell diversity and network dynamics in J.A. Knoblich, R. Jaenisch, Induction of expansion and folding in human cerebral photosensitive human brain organoids, Nature 545 (7652) (2017) 48–53. organoids, Cell Stem Cell 20 (3) (2017) 385–396 e3. [10] M.A. Lancaster, M. Renner, C.A. Martin, D. Wenzel, L.S. Bicknell, M.E. Hurles, [31] E. Karzbrun, A. Kshirsagar, S.R. Cohen, J.H. Hanna, O. Reiner, Human brain or- T. Homfray, J.M. Penninger, A.P. Jackson, J.A. Knoblich, Cerebral organoids ganoids on a chip reveal the physics of folding, Nat. Phys. 14 (5) (2018) 515–522. model human brain development and microcephaly, Nature 501 (7467) (2013) [32] A.C. O’Neill, C. Kyrousi, J. Klaus, R.J. Leventer, E.P. Kirk, A. Fry, D.T. Pilz, 373–379. T. Morgan, Z.A. Jenkins, M. Drukker, S.F. Berkovic, I.E. Scheffer, R. Guerrini, [11] X. Qian, H.N. Nguyen, M.M. Song, C. Hadiono, S.C. Ogden, C. Hammack, B. Yao, D.M. Markie, M. Gotz, S. Cappello, S.P. Robertson, A primate-specific isoform of G.R. Hamersky, F. Jacob, C. Zhong, K.J. Yoon, W. Jeang, L. Lin, Y. Li, J. Thakor, PLEKHG6 regulates neurogenesis and neuronal migration, Cell Rep. 25 (10) D.A. Berg, C. Zhang, E. Kang, M. Chickering, D. Nauen, C.Y. Ho, Z. Wen, (2018) 2729–2741 e6. K.M. Christian, P.Y. Shi, B.J. Maher, H. Wu, P. Jin, H. Tang, H. Song, G.L. Ming, [33] J. Klaus, S. Kanton, C. Kyrousi, A.C. Ayo-Martin, R. Di Giaimo, S. Riesenberg, Brain-region-specific organoids using mini-bioreactors for modeling ZIKV ex- A.C. O’Neill, J.G. Camp, C. Tocco, M. Santel, E. Rusha, M. Drukker, M. Schroeder, posure, Cell 165 (5) (2016) 1238–1254. M. Gotz, S.P. Robertson, B. Treutlein, S. Cappello, Altered neuronal migratory [12] H. Sakaguchi, T. Kadoshima, M. Soen, N. Narii, Y. Ishida, M. Ohgushi, trajectories in human cerebral organoids derived from individuals with neuronal J. Takahashi, M. Eiraku, Y. Sasai, Generation of functional hippocampal neurons heterotopia, Nat. Med. 25 (4) (2019) 561–568. from self-organizing human -derived dorsomedial tele- [34] S. Cappello, M.J. Gray, C. Badouel, S. Lange, M. Einsiedler, M. Srour, D. Chitayat, ncephalic tissue, Nat. Commun. 6 (2015) 8896. F.F. Hamdan, Z.A. Jenkins, T. Morgan, N. Preitner, T. Uster, J. Thomas, [13] X. Qian, F. Jacob, M.M. Song, H.N. Nguyen, H. Song, G.L. Ming, Generation of P. Shannon, V. Morrison, N. Di Donato, L. Van Maldergem, T. Neuhann, human brain region-specific organoids using a miniaturized spinning bioreactor, R. Newbury-Ecob, M. Swinkells, P. Terhal, L.C. Wilson, P.J.G. Zwijnenburg, Nat. Protoc. 13 (3) (2018) 565–580. A.J. Sutherland-Smith, M.A. Black, D. Markie, J.L. Michaud, M.A. Simpson, [14] S. Velasco, A.J. Kedaigle, S.K. Simmons, A. Nash, M. Rocha, G. Quadrato, S. Mansour, H. McNeill, M. Götz, S.P. Robertson, Mutations in genes encoding the B. Paulsen, L. Nguyen, X. Adiconis, A. Regev, J.Z. Levin, P. Arlotta, Individual cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical devel- brain organoids reproducibly form cell diversity of the human cerebral cortex, opment, Nat. Genet. 45 (11) (2013) 1300–1308. Nature 570 (7762) (2019) 523–527. [35] G. Kerjan, J.G. Gleeson, Genetic mechanisms underlying abnormal neuronal mi- [15] S. Kanton, M.J. Boyle, Z. He, M. Santel, A. Weigert, F. Sanchis-Calleja, P. Guijarro, gration in classical lissencephaly, Trends Genet. 23 (12) (2007) 623–630. L. Sidow, J.S. Fleck, D. Han, Z. Qian, M. Heide, W.B. Huttner, P. Khaitovich, [36] R. Birnbaum, D.R. Weinberger, Genetic insights into the neurodevelopmental S. Paabo, B. Treutlein, J.G. Camp, Organoid single-cell genomic atlas uncovers origins of schizophrenia, Nat. Rev. Neurosci. 18 (12) (2017) 727–740. human-specific features of brain development, Nature 574 (7778) (2019) [37] N.J. Brandon, J.K. Millar, C. Korth, H. Sive, K.K. Singh, A. Sawa, Understanding 418–422. the role of DISC1 in psychiatric disease and during normal development, J. [16] K.J. Yoon, F.R. Ringeling, C. Vissers, F. Jacob, M. Pokrass, D. Jimenez-Cyrus, Neurosci. 29 (41) (2009) 12768–12775. Y. Su, N.S. Kim, Y. Zhu, L. Zheng, S. Kim, X. Wang, L.C. Dore, P. Jin, S. Regot, [38] K.K. Singh, G. De Rienzo, L. Drane, Y. Mao, Z. Flood, J. Madison, M. Ferreira,

8 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx

S. Bergen, C. King, P. Sklar, H. Sive, L.-H. Tsai, Common DISC1 polymorphisms disease-induced pluripotent stem cells exhibit impaired neurodifferentiation, J. disrupt Wnt/GSK3β signaling and brain development, Neuron 72 (4) (2011) Lipid Res. 59 (3) (2018) 550–563. 545–558. [61] M. Madhavan, Z.S. Nevin, H.E. Shick, E. Garrison, C. Clarkson-Paredes, M. Karl, [39] P. Srikanth, K. Han, Dana G. Callahan, E. Makovkina, Christina R. Muratore, B.L.L. Clayton, D.C. Factor, K.C. Allan, L. Barbar, T. Jain, P. Douvaras, V. Fossati, Matthew A. Lalli, H. Zhou, Justin D. Boyd, Kenneth S. Kosik, Dennis J. Selkoe, R.H. Miller, P.J. Tesar, Induction of myelinating oligodendrocytes in human cor- Tracy L. Young-Pearse, Genomic < em > DISC1 < /em > disruption in hiPSCs tical spheroids, Nat. Methods 15 (9) (2018) 700–706. alters wnt signaling and neural cell fate, Cell Rep. 12 (9) (2015) 1414–1429. [62] M.J. Gorman, E.A. Caine, K. Zaitsev, M.C. Begley, J. Weger-Lucarelli, [40] P. Srikanth, V.N. Lagomarsino, C.R. Muratore, S.C. Ryu, A. He, W.M. Taylor, M.B. Uccellini, S. Tripathi, J. Morrison, B.L. Yount, K.H. Dinnon 3rd, C. Rückert, C. Zhou, M. Arellano, T.L. Young-Pearse, Shared effects of DISC1 disruption and M.C. Young, Z. Zhu, S.J. Robertson, K.L. McNally, J. Ye, B. Cao, I.U. Mysorekar, elevated WNT signaling in human cerebral organoids, Transl. Psychiatry 8 (1) G.D. Ebel, R.S. Baric, S.M. Best, M.N. Artyomov, A. Garcia-Sastre, M.S. Diamond, (2018) 77. An immunocompetent mouse model of zika virus infection, Cell Host Microbe 23 [41] N.A. Sachs, A. Sawa, S.E. Holmes, C.A. Ross, L.E. DeLisi, R.L. Margolis, A frame- (5) (2018) 672–685 e6. shift mutation in disrupted in schizophrenia 1 in an American family with schi- [63] H. Takaki, H. Oshiumi, M. Shingai, M. Matsumoto, T. Seya, Development of mouse zophrenia and schizoaffective disorder, Mol. Psychiatry 10 (8) (2005) 758–764. models for analysis of human virus infections, Microbiol. Immunol. 61 (3-4) [42] F. Ye, E. Kang, C. Yu, X. Qian, F. Jacob, C. Yu, M. Mao, R.Y.C. Poon, J. Kim, (2017) 107–113. H. Song, G.L. Ming, M. Zhang, DISC1 regulates neurogenesis via modulating ki- [64] G.L. Ming, H. Tang, H. Song, Advances in zika virus research: stem cell models, netochore attachment of Ndel1/Nde1 during mitosis, Neuron 96 (5) (2017) challenges, and opportunities, Cell Stem Cell 19 (6) (2016) 690–702. 1041–1054 e5. [65] Z. Wen, H. Song, G.L. Ming, How does zika virus cause microcephaly? Genes Dev. [43] R. Stoner, M.L. Chow, M.P. Boyle, S.M. Sunkin, P.R. Mouton, S. Roy, A. Wynshaw- 31 (9) (2017) 849–861. Boris, S.A. Colamarino, E.S. Lein, E. Courchesne, Patches of disorganization in the [66] X. Qian, H.N. Nguyen, F. Jacob, H. Song, G.L. Ming, Using brain organoids to neocortex of children with autism, N. Engl. J. Med. 370 (13) (2014) 1209–1219. understand zika virus-induced microcephaly, Development 144 (6) (2017) [44] M. Johnstone, N.A. Vasistha, M.C. Barbu, O. Dando, K. Burr, E. Christopher, 952–957. S. Glen, C. Robert, R. Fetit, K.G. Macleod, M.R. Livesey, D.S. Clair, [67] P.P. Garcez, E.C. Loiola, R. Madeiro da Costa, L.M. Higa, P. Trindade, D.H.R. Blackwood, K. Millar, N.O. Carragher, G.E. Hardingham, D.J.A. Wyllie, R. Delvecchio, J.M. Nascimento, R. Brindeiro, A. Tanuri, S.K. Rehen, Zika virus E.C. Johnstone, H.C. Whalley, A.M. McIntosh, S.M. Lawrie, S. Chandran, Reversal impairs growth in human neurospheres and brain organoids, Science 352 (6287) of proliferation deficits caused by chromosome 16p13.11 microduplication (2016) 816–818. through targeting NFkappaB signaling: an integrated study of patient-derived [68] F.R. Cugola, I.R. Fernandes, F.B. Russo, B.C. Freitas, J.L. Dias, K.P. Guimaraes, neuronal precursor cells, cerebral organoids and in vivo brain imaging, Mol. C. Benazzato, N. Almeida, G.C. Pignatari, S. Romero, C.M. Polonio, I. Cunha, Psychiatry 24 (2) (2019) 294–311. C.L. Freitas, W.N. Brandao, C. Rossato, D.G. Andrade, P. Faria Dde, A.T. Garcez, [45] E.K. Stachowiak, C.A. Benson, S.T. Narla, A. Dimitri, L.E.B. Chuye, S. Dhiman, C.A. Buchpigel, C.T. Braconi, E. Mendes, A.A. Sall, P.M. Zanotto, J.P. Peron, K. Harikrishnan, S. Elahi, D. Freedman, K.J. Brennand, P. Sarder, A.R. Muotri, P.C. Beltrao-Braga, The Brazilian zika virus strain causes birth defects M.K. Stachowiak, Cerebral organoids reveal early cortical maldevelopment in in experimental models, Nature 534 (7606) (2016) 267–271. schizophrenia-computational anatomy and genomics, role of FGFR1, Transl. [69] T.J. Nowakowski, A.A. Pollen, E. Di Lullo, C. Sandoval-Espinosa, M. Bershteyn, Psychiatry 7 (11) (2017) 6. A.R. Kriegstein, Expression analysis highlights AXL as a candidate zika virus entry [46] J.W. Lunden, M. Durens, A.W. Phillips, M.W. Nestor, Cortical interneuron function receptor in neural stem cells, Cell Stem Cell 18 (5) (2016) 591–596. in autism spectrum condition, Pediatr. Res. 85 (2) (2019) 146–154. [70] M.F. Wells, M.R. Salick, O. Wiskow, D.J. Ho, K.A. Worringer, R.J. Ihry, [47] J. Mariani, G. Coppola, P. Zhang, A. Abyzov, L. Provini, L. Tomasini, S. Kommineni, B. Bilican, J.R. Klim, E.J. Hill, L.T. Kane, C. Ye, A. Kaykas, M. Amenduni, A. Szekely, D. Palejev, M. Wilson, M. Gerstein, E.L. Grigorenko, K. Eggan, Genetic ablation of AXL does not protect human neural progenitor cells K. Chawarska, K.A. Pelphrey, J.R. Howe, F.M. Vaccarino, FOXG1-dependent and cerebral organoids from zika virus infection, Cell Stem Cell 19 (6) (2016) dysregulation of GABA/Glutamate neuron differentiation in autism spectrum 703–708. disorders, Cell 162 (2) (2015) 375–390. [71] J. Muffat, Y. Li, A. Omer, A. Durbin, I. Bosch, G. Bakiasi, E. Richards, A. Meyer, [48] P. Wang, R. Mokhtari, E. Pedrosa, M. Kirschenbaum, C. Bayrak, D. Zheng, L. Gehrke, R. Jaenisch, Human induced pluripotent stem cell-derived glial cells H.M. Lachman, CRISPR/Cas9-mediated heterozygous knockout of the autism gene and neural progenitors display divergent responses to Zika and dengue infections, CHD8 and characterization of its transcriptional networks in cerebral organoids Proc. Natl. Acad. Sci. U. S. A. 115 (27) (2018) 7117–7122. derived from iPS cells, Mol. Autism 8 (2017) 11. [72] N.V. Ayala-Nunez, G. Follain, F. Delalande, A. Hirschler, E. Partiot, G.L. Hale, [49] F. Birey, J. Andersen, C.D. Makinson, S. Islam, W. Wei, N. Huber, H.C. Fan, B.C. Bollweg, J. Roels, M. Chazal, F. Bakoa, M. Carocci, S. Bourdoulous, K.R.C. Metzler, G. Panagiotakos, N. Thom, N.A. O’Rourke, L.M. Steinmetz, O. Faklaris, S.R. Zaki, A. Eckly, B. Uring-Lambert, F. Doussau, S. Cianferani, J.A. Bernstein, J. Hallmayer, J.R. Huguenard, S.P. Pasca, Assembly of functionally C. Carapito, F.M.J. Jacobs, N. Jouvenet, J.G. Goetz, R. Gaudin, Zika virus enhances integrated human forebrain spheroids, Nature 545 (7652) (2017) 54–59. monocyte adhesion and transmigration favoring viral dissemination to neural [50] L. Chakrabarti, T.K. Best, N.P. Cramer, R.S.E. Carney, J.T.R. Isaac, Z. Galdzicki, cells, Nat. Commun. 10 (1) (2019) 4430. T.F. Haydar, Olig1 and Olig2 triplication causes developmental brain defects in [73] E. Gabriel, A. Ramani, U. Karow, M. Gottardo, K. Natarajan, L.M. Gooi, G. Goranci- down syndrome, Nat. Neurosci. 13 (8) (2010) 927–934. Buzhala, O. Krut, F. Peters, M. Nikolic, S. Kuivanen, E. Korhonen, T. Smura, [51] D.H. Meijer, M.F. Kane, S. Mehta, H. Liu, E. Harrington, C.M. Taylor, C.D. Stiles, O. Vapalahti, A. Papantonis, J. Schmidt-Chanasit, M. Riparbelli, G. Callaini, D.H. Rowitch, Separated at birth? The functional and molecular divergence of M. Kronke, O. Utermohlen, J. Gopalakrishnan, Recent zika virus isolates induce OLIG1 and OLIG2, nature reviews, Neuroscience 13 (12) (2012) 819–831. premature differentiation of neural progenitors in human brain organoids, Cell [52] A. Bhattacharyya, E. McMillan, S.I. Chen, K. Wallace, C.N. Svendsen, A critical Stem Cell 20 (3) (2017) 397–406 e5. period in cortical interneuron neurogenesis in down syndrome revealed by human [74] K.J. Yoon, G. Song, X. Qian, J. Pan, D. Xu, H.S. Rho, N.S. Kim, C. Habela, L. Zheng, neural progenitor cells, Dev. Neurosci. 31 (6) (2009) 497–510. F. Jacob, F. Zhang, E.M. Lee, W.K. Huang, F.R. Ringeling, C. Vissers, C. Li, L. Yuan, [53] J. Lu, G. Lian, H. Zhou, G. Esposito, L. Steardo, L.C. Delli-Bovi, J.L. Hecht, Q.R. Lu, K. Kang, S. Kim, J. Yeo, Y. Cheng, S. Liu, Z. Wen, C.F. Qin, Q. Wu, K.M. Christian, V. Sheen, OLIG2 over-expression impairs proliferation of human down syndrome H. Tang, P. Jin, Z. Xu, J. Qian, H. Zhu, H. Song, G.L. Ming, Zika-virus-encoded neural progenitors, Hum. Mol. Genet. 21 (10) (2012) 2330–2340. NS2A disrupts mammalian cortical neurogenesis by degrading adherens Junction [54] I. Jakovcevski, N. Zecevic, Olig transcription factors are expressed in oligoden- proteins, Cell Stem Cell 21 (3) (2017) 349–358 e6. drocyte and neuronal cells in human fetal CNS, J. Neurosci. 25 (44) (2005) [75] J. Dang, S.K. Tiwari, G. Lichinchi, Y. Qin, V.S. Patil, A.M. Eroshkin, T.M. Rana, 10064–10073. Zika virus depletes neural progenitors in human cerebral organoids through ac- [55] M.A. Petryniak, G.B. Potter, D.H. Rowitch, J.L.R. Rubenstein, Dlx1 and Dlx2 tivation of the innate immune receptor TLR3, Cell Stem Cell 19 (2) (2016) control neuronal versus oligodendroglial cell fate acquisition in the developing 258–265. forebrain, Neuron 55 (3) (2007) 417–433. [76] K.J. Yoon, G. Song, X. Qian, J. Pan, D. Xu, H.S. Rho, N.S. Kim, C. Habela, L. Zheng, [56] Q.R. Lu, D.-i. Yuk, J.A. Alberta, Z. Zhu, I. Pawlitzky, J. Chan, A.P. McMahon, F. Jacob, F. Zhang, E.M. Lee, W.K. Huang, F.R. Ringeling, C. Vissers, C. Li, L. Yuan, C.D. Stiles, D.H. Rowitch, Sonic Hedgehog–Regulated oligodendrocyte lineage K. Kang, S. Kim, J. Yeo, Y. Cheng, S. Liu, Z. Wen, C.F. Qin, Q. Wu, K.M. Christian, genes encoding bHLH proteins in the mammalian , Neuron H. Tang, P. Jin, Z. Xu, J. Qian, H. Zhu, H. Song, G.L. Ming, Zika-virus-encoded 25 (2) (2000) 317–329. NS2A disrupts mammalian cortical neurogenesis by degrading adherens Junction [57] R. Xu, A.T. Brawner, S. Li, J.J. Liu, H. Kim, H. Xue, Z.P. Pang, W.Y. Kim, R.P. Hart, proteins, Cell Stem Cell (2017). Y. Liu, P. Jiang, OLIG2 drives abnormal neurodevelopmental phenotypes in [77] S. Janssens, M. Schotsaert, R. Karnik, V. Balasubramaniam, M. Dejosez, human iPSC-based organoid and chimeric mouse models of Down syndrome, Cell A. Meissner, A. Garcia-Sastre, T.P. Zwaka, Zika virus alters DNA methylation of Stem Cell 24 (6) (2019) 908–926 e8. neural genes in an organoid model of the developing human brain, mSystems 3 (1) [58] N. Mellios, D.A. Feldman, S.D. Sheridan, J.P.K. Ip, S. Kwok, S.K. Amoah, B. Rosen, (2018). B.A. Rodriguez, B. Crawford, R. Swaminathan, S. Chou, Y. Li, M. Ziats, C. Ernst, [78] M. Xu, E.M. Lee, Z. Wen, Y. Cheng, W.K. Huang, X. Qian, J. Tcw, J. Kouznetsova, R. Jaenisch, S.J. Haggarty, M. Sur, MeCP2-regulated miRNAs control early human S.C. Ogden, C. Hammack, F. Jacob, H.N. Nguyen, M. Itkin, C. Hanna, P. Shinn, neurogenesis through differential effects on ERK and AKT signaling, Mol. C. Allen, S.G. Michael, A. Simeonov, W. Huang, K.M. Christian, A. Goate, Psychiatry 23 (4) (2018) 1051–1065. K.J. Brennand, R. Huang, M. Xia, G.L. Ming, W. Zheng, H. Song, H. Tang, [59] Y.L. Latour, R. Yoon, S.E. Thomas, C. Grant, C. Li, M. Sena-Esteves, M.L. Allende, Identification of small-molecule inhibitors of zika virus infection and induced R.L. Proia, C.J. Tifft, Human GLB1 knockout cerebral organoids: a model system neural cell death via a drug repurposing screen, Nat. Med. 22 (10) (2016) for testing AAV9-mediated GLB1 gene therapy for reducing GM1 ganglioside 1101–1107. storage in GM1 gangliosidosis, Mol. Genet. Metab. Rep. 21 (2019) 100513. [79] T. Zhou, L. Tan, G.Y. Cederquist, Y. Fan, B.J. Hartley, S. Mukherjee, [60] M.L. Allende, E.K. Cook, B.C. Larman, A. Nugent, J.M. Brady, D. Golebiowski, M. Tomishima, K.J. Brennand, Q. Zhang, R.E. Schwartz, T. Evans, L. Studer, M. Sena-Esteves, C.J. Tifft, R.L. Proia, Cerebral organoids derived from sandhoff S. Chen, High-content screening in hPSC-neural progenitors identifies drug

9 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx

candidates that inhibit zika virus infection in fetal-like organoids and adult brain, minocycline and rapamycin in gamma-irradiated human embryonic stem cells- Cell Stem Cell 21 (2) (2017) 274–283 e5. derived cerebral organoids, Mol. Biol. Rep. 46 (1) (2019) 1343–1348. [80] C.Q. Sacramento, G.R. de Melo, C.S. de Freitas, N. Rocha, L.V. Hoelz, M. Miranda, [105] M.P. Schwartz, Z. Hou, N.E. Propson, J. Zhang, C.J. Engstrom, V. Santos Costa, N. Fintelman-Rodrigues, A. Marttorelli, A.C. Ferreira, G. Barbosa-Lima, P. Jiang, B.K. Nguyen, J.M. Bolin, W. Daly, Y. Wang, R. Stewart, C.D. Page, J.L. Abrantes, Y.R. Vieira, M.M. Bastos, E. de Mello Volotao, E.P. Nunes, W.L. Murphy, J.A. Thomson, Human pluripotent stem cell-derived neural con- D.A. Tschoeke, L. Leomil, E.C. Loiola, P. Trindade, S.K. Rehen, F.A. Bozza, structs for predicting neural toxicity, Proc. Natl. Acad. Sci. U. S. A. 112 (40) (2015) P.T. Bozza, N. Boechat, F.L. Thompson, A.M. de Filippis, K. Bruning, T.M. Souza, 12516–12521. The clinically approved antiviral drug sofosbuvir inhibits zika virus replication, [106] S.H. Choi, Y.H. Kim, M. Hebisch, C. Sliwinski, S. Lee, C. D’Avanzo, H. Chen, Sci. Rep. 7 (2017) 40920. B. Hooli, C. Asselin, J. Muffat, J.B. Klee, C. Zhang, B.J. Wainger, M. Peitz, [81] M. Watanabe, J.E. Buth, N. Vishlaghi, L. de la Torre-Ubieta, J. Taxidis, B.S. Khakh, D.M. Kovacs, C.J. Woolf, S.L. Wagner, R.E. Tanzi, D.Y. Kim, A three-dimensional G. Coppola, C.A. Pearson, K. Yamauchi, D. Gong, X. Dai, R. Damoiseaux, human neural cell culture model of alzheimer’s disease, Nature 515 (7526) (2014) R. Aliyari, S. Liebscher, K. Schenke-Layland, C. Caneda, E.J. Huang, Y. Zhang, 274–278. G. Cheng, D.H. Geschwind, P. Golshani, R. Sun, B.G. Novitch, Self-organized [107] A.M. Hall, E.D. Roberson, Mouse models of alzheimer’s disease, Brain Res. Bull. 88 cerebral organoids with human-specific features predict effective drugs to combat (1) (2012) 3–12. zika virus infection, Cell Rep. 21 (2) (2017) 517–532. [108] G.A. Elder, M.A. Gama Sosa, R. De Gasperi, Transgenic mouse models of alzhei- [82] C. Li, Y.Q. Deng, S. Wang, F. Ma, R. Aliyari, X.Y. Huang, N.N. Zhang, M. Watanabe, mer’s disease, Mt. Sinai J. Med. 77 (1) (2010) 69–81. H.L. Dong, P. Liu, X.F. Li, Q. Ye, M. Tian, S. Hong, J. Fan, H. Zhao, L. Li, [109] B.L. Goode, M. Chau, P.E. Denis, S.C. Feinstein, Structural and functional differ- N. Vishlaghi, J.E. Buth, C. Au, Y. Liu, N. Lu, P. Du, F.X. Qin, B. Zhang, D. Gong, ences between 3-repeat and 4-repeat tau isoforms: IMPLICATIONS FOR NORMAL X. Dai, R. Sun, B.G. Novitch, Z. Xu, C.F. Qin, G. Cheng, 25-hydroxycholesterol tau function and the onset of neurodegenerative disease, J. Biol. Chem. 275 (49) protects host against zika virus infection and its associated microcephaly in a (2000) 38182–38189. mouse model, Immunity 46 (3) (2017) 446–456. [110] E. Rockenstein, C.R. Overk, K. Ubhi, M. Mante, C. Patrick, A. Adame, A. Bisquert, [83] Y.P. Xu, Y. Qiu, B. Zhang, G. Chen, Q. Chen, M. Wang, F. Mo, J. Xu, J. Wu, M. Trejo-Morales, B. Spencer, E. Masliah, A novel triple repeat mutant tau trans- R.R. Zhang, M.L. Chen, N.N. Zhang, B. Lyu, W.L. Zhu, M.H. Wu, Q. Ye, D. Zhang, genic model that mimics aspects of pick’s disease and fronto-temporal tauopathies, J.H. Man, X.F. Li, J. Cui, Z. Xu, B. Hu, X. Zhou, C.F. Qin, Zika virus infection PLoS One 10 (3) (2015) e0121570-e0121570. induces RNAi-mediated antiviral immunity in human neural progenitors and brain [111] S. Salloway, R. Sperling, N.C. Fox, K. Blennow, W. Klunk, M. Raskind, M. Sabbagh, organoids, Cell Res. 29 (4) (2019) 265–273. L.S. Honig, A.P. Porsteinsson, S. Ferris, M. Reichert, N. Ketter, B. Nejadnik, [84] B. Zhang, Y. He, Y. Xu, F. Mo, T. Mi, Q.S. Shen, C. Li, Y. Li, J. Liu, Y. Wu, G. Chen, V. Guenzler, M. Miloslavsky, D. Wang, Y. Lu, J. Lull, I.C. Tudor, E. Liu, W. Zhu, C. Qin, B. Hu, G. Zhou, Differential antiviral immunity to Japanese en- M. Grundman, E. Yuen, R. Black, H.R. Brashear, Two phase 3 trials of bapineu- cephalitis virus in developing cortical organoids, Cell Death Dis. 9 (7) (2018) 719. zumab in mild-to-moderate alzheimer’s disease, N. Engl. J. Med. 370 (4) (2014) [85] M.C. Cheeran, J.R. Lokensgard, M.R. Schleiss, Neuropathogenesis of congenital 322–333. cytomegalovirus infection: disease mechanisms and prospects for intervention, [112] R.S. Doody, R. Raman, M. Farlow, T. Iwatsubo, B. Vellas, S. Joffe, K. Kieburtz, Clin. Microbiol. Rev. 22 (1) (2009) 99–126. Table of Contents. F. He, X. Sun, R.G. Thomas, P.S. Aisen, E. Siemers, G. Sethuraman, R. Mohs, A [86] G. Sun, F. Chiuppesi, X. Chen, C. Wang, E. Tian, J. Nguyen, M. Kha, D. Trinh, phase 3 trial of semagacestat for treatment of alzheimer’s disease, N. Engl. J. Med. H. Zhang, M.C. Marchetto, H. Song, G.-L. Ming, F.H. Gage, D.J. Diamond, 369 (4) (2013) 341–350. F. Wussow, Y. Shi, Modeling human cytomegalovirus-induced microcephaly in [113] R.S. Doody, R.G. Thomas, M. Farlow, T. Iwatsubo, B. Vellas, S. Joffe, K. Kieburtz, human iPSC-derived brain organoids, Cell Rep. Med. 1 (1) (2020) 100002. R. Raman, X. Sun, P.S. Aisen, E. Siemers, H. Liu-Seifert, R. Mohs, Phase 3 trials of [87] S.L. Sison, B.S. O’Brien, A.J. Johnson, E.R. Seminary, S.S. Terhune, A.D. Ebert, solanezumab for mild-to-moderate alzheimer’s disease, N. Engl. J. Med. 370 (4) Human cytomegalovirus disruption of calcium signaling in neural progenitor cells (2014) 311–321. and organoids, J. Virol. 93 (17) (2019) e00954–19. [114] W.K. Raja, A.E. Mungenast, Y.T. Lin, T. Ko, F. Abdurrob, J. Seo, L.H. Tsai, Self- [88] L. Wagenführ, A.K. Meyer, L. Braunschweig, L. Marrone, A. Storch, Brain oxygen organizing 3D human neural tissue derived from induced pluripotent stem cells tension controls the expansion of outer subventricular zone-like basal progenitors recapitulate alzheimer’s disease phenotypes, PLoS One 11 (9) (2016) e0161969. in the developing mouse brain, Development 142 (17) (2015) 2904–2915. [115] C. Gonzalez, E. Armijo, J. Bravo-Alegria, A. Becerra-Calixto, C.E. Mays, C. Soto, [89] A.M. Pasca, J.Y. Park, H.W. Shin, Q. Qi, O. Revah, R. Krasnoff,R.O’Hara, Modeling and tau pathology in human cerebral organoids, Mol. A.J. Willsey, T.D. Palmer, S.P. Pasca, Human 3D cellular model of hypoxic brain Psychiatry 23 (12) (2018) 2363–2374. injury of prematurity, Nat. Med. 25 (5) (2019) 784–791. [116] Y. Yan, L. Song, J. Bejoy, J. Zhao, T. Kanekiyo, G. Bu, Y. Zhou, Y. Li, Modeling [90] N. Daviaud, C. Chevalier, R.H. Friedel, H. Zou, Distinct vulnerability and resilience neurodegenerative microenvironment using cortical organoids derived from of human neuroprogenitor subtypes in cerebral organoid model of prenatal hy- human stem cells, Tissue Eng. Part A 24 (13-14) (2018) 1125–1137. poxic injury, Front. Cell. Neurosci. 13 (2019) 336. [117] Y.T. Lin, J. Seo, F. Gao, H.M. Feldman, H.L. Wen, J. Penney, H.P. Cam, [91] E.M. Boisvert, R.E. Means, M. Michaud, J.A. Madri, S.G. Katz, Minocycline miti- E. Gjoneska, W.K. Raja, J. Cheng, R. Rueda, O. Kritskiy, F. Abdurrob, Z. Peng, gates the effect of neonatal hypoxic insult on human brain organoids, Cell Death B. Milo, C.J. Yu, S. Elmsaouri, D. Dey, T. Ko, B.A. Yankner, L.H. Tsai, APOE4 Dis. 10 (4) (2019) 325. causes widespread molecular and cellular alterations associated with alzheimer’s [92] Y. Zhu, L. Wang, F. Yin, Y. Yu, Y. Wang, M.J. Shepard, Z. Zhuang, J. Qin, Probing disease phenotypes in human iPSC-derived brain cell types, Neuron 98 (6) (2018) impaired neurogenesis in human brain organoids exposed to alcohol, Integr. Biol. 1141–1154 e7. (Camb.) 9 (12) (2017) 968–978. [118] K. Meyer, H.M. Feldman, T. Lu, D. Drake, E.T. Lim, K.H. Ling, N.A. Bishop, Y. Pan, [93] C.T. Lee, J. Chen, A.A. Kindberg, R.M. Bendriem, C.E. Spivak, M.P. Williams, J. Seo, Y.T. Lin, S.C. Su, G.M. Church, L.H. Tsai, B.A. Yankner, REST and neural C.T. Richie, A. Handreck, B.S. Mallon, C.R. Lupica, D.T. Lin, B.K. Harvey, Gene network dysregulation in iPSC models of alzheimer’s disease, Cell Rep. 26 (5) D.C. Mash, W.J. Freed, CYP3A5 Mediates Effects of Cocaine on Human (2019) 1112–1127 e9. Neocorticogenesis: Studies using an In Vitro 3D Self-organized hPSC model with a [119] S. Pavoni, R. Jarray, F. Nassor, A.C. Guyot, S. Cottin, J. Rontard, J. Mikol, single cortex-like unit, Neuropsychopharmacology 42 (3) (2017) 774–784. A. Mabondzo, J.P. Deslys, F. Yates, Small-molecule induction of abeta-42 peptide [94] Y. Wang, L. Wang, Y. Zhu, J. Qin, Human brain organoid-on-a-chip to model production in human cerebral organoids to model alzheimer’s disease associated prenatal nicotine exposure, Lab Chip 18 (6) (2018) 851–860. phenotypes, PLoS One 13 (12) (2018) e0209150. [95] V. Dakic, J. Minardi Nascimento, R. Costa Sartore, R.M. Maciel, D.B. de Araujo, [120] Y. Gerakis, C. Hetz, Brain organoids: a next step for humanized alzheimer’s disease S. Ribeiro, D. Martins-de-Souza, S.K. Rehen, Short term changes in the proteome of models? Mol. Psychiatry 24 (4) (2019) 474–478. human cerebral organoids induced by 5-MeO-DMT, Sci. Rep. 7 (1) (2017) 12863. [121] J. Seo, O. Kritskiy, L.A. Watson, S.J. Barker, D. Dey, W.K. Raja, Y.T. Lin, T. Ko, [96] S.J. Stock, J.E. Norman, Medicines in pregnancy, F1000Res 8 (2019). S. Cho, J. Penney, M.C. Silva, S.D. Sheridan, D. Lucente, J.F. Gusella, [97] B.L. Thompson, P. Levitt, G.D. Stanwood, Prenatal exposure to drugs: effects on B.C. Dickerson, S.J. Haggarty, L.H. Tsai, Inhibition of p25/Cdk5 attenuates tauo- brain development and implications for policy and education, Nat. Rev. Neurosci. pathy in mouse and iPSC models of frontotemporal dementia, J. Neurosci. 37 (41) 10 (4) (2009) 303–312. (2017) 9917–9924. [98] E.A. Bonney, Demystifying animal models of adverse pregnancy outcomes: [122] W. Noble, V. Olm, K. Takata, E. Casey, O. Mary, J. Meyerson, K. Gaynor, touching bench and bedside, Am. J. Reprod. Immunol. 69 (6) (2013) 567–584. J. LaFrancois, L. Wang, T. Kondo, P. Davies, M. Burns, Veeranna, R. Nixon, [99] N. Vargesson, Thalidomide-induced teratogenesis: history and mechanisms, Birth D. Dickson, Y. Matsuoka, M. Ahlijanian, L.-F. Lau, K. Duff, Cdk5 Is a key factor in Defects Res. Part C: Today: Rev. 105 (2) (2015) 140–156. tau aggregation and tangle formation in vivo, Neuron 38 (4) (2003) 555–565. [100] I.D. Fratta, E.B. Sigg, K. Maiorana, Teratogenic effects of thalidomide in rabbits, [123] J.C. Cruz, H.-C. Tseng, J.A. Goldman, H. Shih, L.-H. Tsai, Aberrant Cdk5 activation rats, hamsters, and mice, Toxicol. Appl. Pharmacol. 7 (2) (1965) 268–286. by p25 triggers pathological events leading to and neurofi- [101] F. Liu, J. Huang, Z. Liu, Vincristine impairs microtubules and causes neurotoxicity brillary tangles, Neuron 40 (3) (2003) 471–483. in cerebral organoids, Neuroscience 404 (2019) 530–540. [124] P. Conforti, D. Besusso, V.D. Bocchi, A. Faedo, E. Cesana, G. Rossetti, V. Ranzani, [102] J. Huang, F. Liu, H. Tang, H. Wu, L. Li, R. Wu, J. Zhao, Y. Wu, Z. Liu, J. Chen, C.N. Svendsen, L.M. Thompson, M. Toselli, G. Biella, M. Pagani, E. Cattaneo, Tranylcypromine causes neurotoxicity and represses BHC110/LSD1 in human-in- Faulty neuronal determination and cell polarization are reverted by modulating duced pluripotent stem cell-derived cerebral organoids model, Front. Neurol. 8 HD early phenotypes, Proc. Natl. Acad. Sci. U. S. A. 115 (4) (2018) E762–E771. (2017) 626. [125] B.R. Groveman, S.T. Foliaki, C.D. Orru, G. Zanusso, J.A. Carroll, B. Race, [103] Z. Hubler, D. Allimuthu, I. Bederman, M.S. Elitt, M. Madhavan, K.C. Allan, C.L. Haigh, Sporadic creutzfeldt-jakob disease prion infection of human cerebral H.E. Shick, E. Garrison, T.K. M, D.C. Factor, Z.S. Nevin, J.L. Sax, M.A. Thompson, organoids, Acta Neuropathol. Commun. 7 (1) (2019) 90. Y. Fedorov, J. Jin, W.K. Wilson, M. Giera, F. Bracher, R.H. Miller, P.J. Tesar, [126] F. Perez-Branguli, I.Y. Buchsbaum, T. Pozner, M. Regensburger, W. Fan, A. Schray, D.J. Adams, Accumulation of 8,9-unsaturated sterols drives oligodendrocyte for- T. Borstler, H. Mishra, D. Graf, Z. Kohl, J. Winkler, B. Berninger, S. Cappello, mation and remyelination, Nature 560 (7718) (2018) 372–376. B. Winner, Human SPG11 cerebral organoids reveal cortical neurogenesis im- [104] A. Shakhbazau, N. Danilkovich, I. Seviaryn, T. Ermilova, S. Kosmacheva, Effects of pairment, Hum. Mol. Genet. 28 (6) (2019) 961–971.

10 D.Y. Zhang, et al. Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx

[127] P.B. Crino, Evolving neurobiology of tuberous sclerosis complex, Acta [140] C. Yi, G. Li, D.N. Ivanov, Z. Wang, M.X. Velasco, G. Hernandez, S. Kaundal, Neuropathol. 125 (3) (2013) 317–332. J. Villarreal, Y.K. Gupta, M. Qiao, C.G. Hubert, M.J. Hart, L.O.F. Penalva, Luteolin [128] X. Wu, A. Sosunov, R. Tikoo, H. Weiner, P. Crino, G. McKhann, Glutamate inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma transport is impaired in the human tuberous sclerosis tissue: 3.016, Epilepsia 46 cells, RNA Biol. 15 (11) (2018) 1420–1432. (2005). [141] R. Tejero, Y. Huang, I. Katsyv, M. Kluge, J.Y. Lin, J. Tome-Garcia, N. Daviaud, [129] E.J. Uhlmann, M. Wong, R.L. Baldwin, M.L. Bajenaru, H. Onda, D.J. Kwiatkowski, Y. Wang, B. Zhang, N.M. Tsankova, C.C. Friedel, H. Zou, R.H. Friedel, Gene sig- K. Yamada, D.H. Gutmann, Astrocyte-specific TSC1 conditional knockout mice natures of quiescent glioblastoma cells reveal mesenchymal shift and interactions exhibit abnormal neuronal organization and seizures, Ann. Neurol. 52 (3) (2002) with niche microenvironment, EBioMedicine 42 (2019) 252–269. 285–296. [142] F. Jacob, R.D. Salinas, D.Y. Zhang, P.T.T. Nguyen, J.G. Schnoll, S.Z.H. Wong, [130] S.W. Way, J. McKenna 3rd, U. Mietzsch, R.M. Reith, H.C. Wu, M.J. Gambello, Loss R. Thokala, S. Sheikh, D. Saxena, S. Prokop, D.A. Liu, X. Qian, D. Petrov, T. Lucas, of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in H.I. Chen, J.F. Dorsey, K.M. Christian, Z.A. Binder, M. Nasrallah, S. Brem, the mouse, Hum. Mol. Genet. 18 (7) (2009) 1252–1265. D.M. O’Rourke, G.L. Ming, H. Song, A patient-derived glioblastoma organoid [131] L.H. Zeng, N.R. Rensing, B. Zhang, D.H. Gutmann, M.J. Gambello, M. Wong, Tsc2 model and biobank recapitulates inter- and intra-tumoral heterogeneity, Cell 180 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation (1) (2020) 188–204 e22. in a mouse model of tuberous sclerosis complex, Hum. Mol. Genet. 20 (3) (2011) [143] F. Jacob, G.-l. Ming, H. Song, Generation and biobanking of patient-derived 445–454. glioblastoma organoids and their application in CAR- testing, bioRxiv (2020) [132] L. Meikle, D.M. Talos, H. Onda, K. Pollizzi, A. Rotenberg, M. Sahin, F.E. Jensen, 2020.03.19.999110. D.J. Kwiatkowski, A mouse model of tuberous sclerosis: neuronal loss of Tsc1 [144] D.M. van Pel, K. Harada, D. Song, C.C. Naus, W.C. Sin, Modelling glioma invasion causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and using 3D bioprinting and scaffold-free 3D culture, J. Cell Commun. Signal. 12 (4) limited survival, J. Neurosci. 27 (21) (2007) 5546–5558. (2018) 723–730. [133] M. Wong, A tuber-ful animal model of tuberous sclerosis at last? Epilepsy Curr. 12 [145] B. da Silva, R.K. Mathew, E.S. Polson, J. Williams, H. Wurdak, Spontaneous (1) (2012) 15–16. glioblastoma spheroid infiltration of early-stage cerebral organoids models brain [134] D.M. Feliciano, T. Su, J. Lopez, J.C. Platel, A. Bordey, Single-cell Tsc1 knockout tumor invasion, SLAS Discov. 23 (8) (2018) 862–868. during corticogenesis generates tuber-like lesions and reduces seizure threshold in [146] A. Linkous, D. Balamatsias, M. Snuderl, L. Edwards, K. Miyaguchi, T. Milner, mice, J. Clin. Invest. 121 (4) (2011) 1596–1607. B. Reich, L. Cohen-Gould, A. Storaska, Y. Nakayama, E. Schenkein, R. Singhania, [135] J.D. Blair, D. Hockemeyer, H.S. Bateup, Genetically engineered human cortical S. Cirigliano, T. Magdeldin, Y. Lin, G. Nanjangud, K. Chadalavada, D. Pisapia, spheroid models of tuberous sclerosis, Nat. Med. 24 (10) (2018) 1568–1578. C. Liston, H.A. Fine, Modeling patient-derived glioblastoma with cerebral orga- [136] J. Ogawa, G.M. Pao, M.N. Shokhirev, I.M. Verma, Glioblastoma model using noids, Cell Rep. 26 (12) (2019) 3203–3211 e5. human cerebral organoids, Cell Rep. 23 (4) (2018) 1220–1229. [147] A.R. Pine, S.M. Cirigliano, J.G. Nicholson, Y. Hu, A. Linkous, K. Miyaguchi, [137] S. Bian, M. Repic, Z. Guo, A. Kavirayani, T. Burkard, J.A. Bagley, C. Krauditsch, L. Edwards, R. Singhania, T.H. Schwartz, R. Ramakrishna, D.J. Pisapia, J.A. Knoblich, Genetically engineered cerebral organoids model brain tumor for- M. Snuderl, O. Elemento, H.A. Fine, Tumor microenvironment is critical for the mation, Nat. Methods 15 (8) (2018) 631–639. maintenance of cellular states found in primary , Cancer Discov. [138] C.G. Hubert, M. Rivera, L.C. Spangler, Q. Wu, S.C. Mack, B.C. Prager, M. Couce, (2020) CD-20-0057. R.E. McLendon, A.E. Sloan, J.N. Rich, A Three-Dimensional Organoid Culture [148] Y.X. Setoh, A.A. Amarilla, N.Y.G. Peng, R.E. Griffiths, J. Carrera, M.E. Freney, System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients E. Nakayama, S. Ogawa, D. Watterson, N. Modhiran, F.E. Nanyonga, F.J. Torres, and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo, Cancer Res. 76 (8) A. Slonchak, P. Periasamy, N.A. Prow, B. Tang, J. Harrison, J. Hobson-Peters, (2016) 2465–2477. T. Cuddihy, J. Cooper-White, R.A. Hall, P.R. Young, J.M. Mackenzie, [139] Z. Zhu, M.J. Gorman, L.D. McKenzie, J.N. Chai, C.G. Hubert, B.C. Prager, E. Wolvetang, J.D. Bloom, A. Suhrbier, A.A. Khromykh, Determinants of zika virus E. Fernandez, J.M. Richner, R. Zhang, C. Shan, E. Tycksen, X. Wang, P.Y. Shi, host tropism uncovered by deep mutational scanning, Nat. Microbiol. 4 (5) (2019) M.S. Diamond, J.N. Rich, M.G. Chheda, Zika virus has oncolytic activity against 876–887. glioblastoma stem cells, J. Exp. Med. 214 (10) (2017) 2843–2857.

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